RESUMO
BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.
Assuntos
Proteína BRCA1/deficiência , Neoplasias da Mama/enzimologia , Replicação do DNA , DNA de Neoplasias/biossíntese , Proteases Específicas de Ubiquitina/metabolismo , Neoplasias do Colo do Útero/enzimologia , Animais , Proteína BRCA1/genética , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Sobrevivência Celular , DNA de Neoplasias/genética , Resistência a Medicamentos , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos Nus , Mutação , Desnaturação de Ácido Nucleico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/genética , Ubiquitinação , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Major depressive disorder demonstrated sex differences in prevalence and symptoms, which were more pronounced during adolescence. Yet, research on sex-specific brain network characteristics in adolescent-onset major depressive disorder remains limited. This study investigated sex-specific and nonspecific alterations in resting-state functional connectivity of three core networks (frontoparietal network, salience network, and default mode network) and subcortical networks in adolescent-onset major depressive disorder, using seed-based resting-state functional connectivity in 50 medication-free patients with adolescent-onset major depressive disorder and 56 healthy controls. Irrespective of sex, compared with healthy controls, adolescent-onset major depressive disorder patients showed hypoconnectivity between bilateral hippocampus and right superior temporal gyrus (default mode network). More importantly, we further found that females with adolescent-onset major depressive disorder exhibited hypoconnectivity within the default mode network (medial prefrontal cortex), and between the subcortical regions (i.e. amygdala, striatum, and thalamus) with the default mode network (angular gyrus and posterior cingulate cortex) and the frontoparietal network (dorsal prefrontal cortex), while the opposite patterns of resting-state functional connectivity alterations were observed in males with adolescent-onset major depressive disorder, relative to their sex-matched healthy controls. Moreover, several sex-specific resting-state functional connectivity changes were correlated with age of onset, sleep disturbance, and anxiety in adolescent-onset major depressive disorder with different sex. These findings suggested that these sex-specific resting-state functional connectivity alterations may reflect the differences in brain development or processes related to early illness onset, underscoring the necessity for sex-tailored diagnostic and therapeutic approaches in adolescent-onset major depressive disorder.
Assuntos
Encéfalo , Transtorno Depressivo Maior , Imageamento por Ressonância Magnética , Rede Nervosa , Caracteres Sexuais , Humanos , Transtorno Depressivo Maior/fisiopatologia , Transtorno Depressivo Maior/diagnóstico por imagem , Feminino , Adolescente , Masculino , Encéfalo/fisiopatologia , Encéfalo/diagnóstico por imagem , Rede Nervosa/diagnóstico por imagem , Rede Nervosa/fisiopatologia , Vias Neurais/fisiopatologia , Vias Neurais/diagnóstico por imagem , Adulto Jovem , Idade de Início , Mapeamento Encefálico , Rede de Modo Padrão/fisiopatologia , Rede de Modo Padrão/diagnóstico por imagemRESUMO
Overexpression of the TGFß pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFß promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFß signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFß pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFß and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFß-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFß-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFß-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis.
Assuntos
Anemia de Fanconi , Camundongos , Animais , Anemia de Fanconi/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: Colorectal cancer (CRC) is a major cause of cancer-related deaths worldwide, and chemoresistance is a major obstacle in its treatment. Despite advances in therapy, the molecular mechanism underlying chemoresistance in CRC is not fully understood. Recent studies have implicated the key roles of long noncoding RNAs (lncRNAs) in the regulation of CRC chemoresistance. METHODS: In this study, we investigated the role of the lncRNA LINC01852 in CRC chemoresistance. LINC01852 expression was evaluated in multiple CRC cohorts using quantitative reverse transcription PCR. We conducted in vitro and in vivo functional experiments using cell culture and mouse models. RNA pull-down, RNA immunoprecipitation, chromatin immunoprecipitation, and dual luciferase assays were used to investigate the molecular mechanism of LINC01852 in CRC. RESULTS: Our findings revealed that a lncRNA with tumor-inhibiting properties, LINC01852, was downregulated in CRC and inhibited cell proliferation and chemoresistance both in vitro and in vivo. Further mechanistic investigations revealed that LINC01852 increases TRIM72-mediated ubiquitination and degradation of SRSF5, inhibiting SRSF5-mediated alternative splicing of PKM and thereby decreasing the production of PKM2. Overexpression of LINC01852 induces a metabolic switch from aerobic glycolysis to oxidative phosphorylation, which attenuates the chemoresistance of CRC cells by inhibiting PKM2-mediated glycolysis. CONCLUSIONS: Our results demonstrate that LINC01852 plays an important role in repressing CRC malignancy and chemoresistance by regulating SRSF5-mediated alternative splicing of PKM, and that targeting the LINC01852/TRIM72/SRSF5/PKM2 signaling axis may represent a potential therapeutic strategy for CRC.
Assuntos
Neoplasias Colorretais , RNA Longo não Codificante , Animais , Camundongos , Humanos , Processamento Alternativo , Resistencia a Medicamentos Antineoplásicos , Carcinogênese , Transformação Celular Neoplásica , Imunoprecipitação da CromatinaRESUMO
Horizontal gene transfer has been demonstrated to be an important driver for the emergency of multidrug-resistant pathogens. Recently, a transferable gene cluster tmexCD1-toprJ1 of the resistance-nodulation-division (RND) superfamily was identified in the plasmids of animal-derived Klebsiella pneumoniae strains, with a higher efflux capacity for various drugs than the Escherichia coli AcrAB-TolC homolog system. In this study, we focused on the differences in the inner membrane pump of these two systems and identified some key residues that contribute to the robust efflux activity of the TMexCD1 system. With the aid of homologous modeling and molecular docking, eight residues from the proximal binding pocket (PBP) and nine from the distal binding pocket (DBP) were selected and subjected to site-directed mutagenesis. Several of them, such as S134, I139, D181, and A290, were shown to be important for substrate binding in the DBP region, and all residues in PBP and DBP showed certain substrate preferences. Apart from the conservative switch loop (L613-623TMexD1) previously identified in the E. coli AcrB (EcAcrB), a relatively unconservative loop (L665-675TMexD1) at the bottom of PBP was proposed as a critical element for the robust activity of TMexD1, due to variations at sites E669, G670, N673, and S674 compared to EcAcrAB, and the significantly altered efflux activity due to their mutations. The conservation and flexibility of these key factors can contribute to the evolution of the RND efflux pumps and thus serve as potential targets for developing inhibitors to block the widespread of the TMexCD1 system.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Antibacterianos/química , Simulação de Acoplamento Molecular , Farmacorresistência Bacteriana Múltipla/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Testes de Sensibilidade MicrobianaRESUMO
In order to avoid the time-consuming and laborious identification of tumor-specific antigens (TSAs) during the traditional vaccine fabrication process, a versatile photodynamic therapy (PDT)-based method is developed to construct a whole-tumor antigen tumor vaccine (TV) from surgically resected tumor tissues for personalized immunotherapy. Mucoadhesive nanoparticles containing small-molecular photosensitizer are fabricated and directly co-incubated with suspended tumor cells obtained after cytoreduction surgery. After irradiation with a 405 nm laser, potent immunogenic cell death of cancer cells could be induced. Along with the release of TSAs, the as-prepared TV could activate safe and robust tumor-specific immune responses, leading to efficient suppression of postsurgery tumor recurrence and metastasis. The as-prepared TV cannot only be applied alone through various administration routes but also synergize with immunoadjuvant, chemotherapeutics, and immune checkpoint blockers to exert more potent immune responses. This work provides an alternative way to promote the clinical translation of PDT, which is generally restricted by the limited penetration of light. Moreover, the versatile strategy of vaccine fabrication also facilitates the clinical application of personalized whole-cell tumor vaccines.
Assuntos
Vacinas Anticâncer , Metástase Neoplásica , Fotoquimioterapia , Fotoquimioterapia/métodos , Vacinas Anticâncer/uso terapêutico , Animais , Humanos , Medicina de Precisão/métodos , Linhagem Celular Tumoral , Recidiva Local de Neoplasia/prevenção & controle , Fármacos Fotossensibilizantes/uso terapêutico , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Camundongos , Nanopartículas/química , Antígenos de Neoplasias/imunologia , Imunoterapia/métodos , FemininoRESUMO
Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.
Assuntos
Proteína BRCA1/deficiência , Dineínas do Citoplasma/metabolismo , DNA/metabolismo , Genes BRCA1 , Proteína Homóloga a MRE11/metabolismo , Reparo de DNA por Recombinação , Proteína BRCA1/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Aberrações Cromossômicas , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Edição de Genes , Instabilidade Genômica/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Humanos , Mutação , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Platina/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica , Reparo de DNA por Recombinação/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Glutamate is one of the most important excitatory neurotransmitters within the mammalian central nervous system. The role of glutamate in regulating neural network signaling transmission through both synaptic and extra-synaptic paths highlights the importance of the real-time and continuous monitoring of its concentration and dynamics in living organisms. Progresses in multidisciplinary research have promoted the development of electrochemical glutamate sensors through the co-design of materials, interfaces, electronic devices, and integrated systems. This review summarizes recent works reporting various electrochemical sensor designs and their applicability as miniaturized neural probes to in vivo sensing within biological environments. We start with an overview of the role and physiological significance of glutamate, the metabolic routes, and its presence in various bodily fluids. Next, we discuss the design principles, commonly employed validation models/protocols, and successful demonstrations of multifunctional, compact, and bio-integrated devices in animal models. The final section provides an outlook on the development of the next generation glutamate sensors for neuroscience and neuroengineering, with the aim of offering practical guidance for future research.
Assuntos
Sistema Nervoso Central , Técnicas Eletroquímicas , Ácido Glutâmico , Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Animais , Sistema Nervoso Central/metabolismo , Humanos , Técnicas Biossensoriais/métodosRESUMO
The hallmark feature of metabolic reprogramming is now considered to be widespread in many malignancies, including colorectal cancer (CRC). Of the gastrointestinal tumors, CRC is one of the most common with a high metastasis rate and long insidious period. The incidence and mortality of CRC has increased in recent years. Metabolic reprogramming also has a significant role in the development and progression of CRC, especially lipid metabolic reprogramming. Many studies have reported that lipid metabolism reprogramming is similar to the Warburg effect with typical features affecting tumor biology including proliferation, migration, local invasion, apoptosis, and other biological behaviors of cancer cells. Therefore, studying the role of lipid metabolism in the occurrence and development of CRC will increase our understanding of its pathogenesis, invasion, metastasis, and other processes and provide new directions for the treatment of CRC. In this paper, we mainly describe the molecular mechanism of lipid metabolism reprogramming and its important role in the occurrence and development of CRC. In addition, to provide reference for subsequent research and clinical diagnosis and treatment we also review the treatments of CRC that target lipid metabolism.
Assuntos
Neoplasias Colorretais , Metabolismo dos Lipídeos , Humanos , Neoplasias Colorretais/metabolismo , Proliferação de Células , Movimento Celular , ApoptoseRESUMO
Sodium-proton (Na+ /H+ ) antiporters from the ion transporter (IT) superfamily play a vital role in controlling the pH and electrolyte homeostasis. However, very limited information regarding their structural functions is available to date. In this study, the structural model of the NhaD antiporter was proposed as a typical hairpin structure of IT proteins, with two symmetrically conserved scaffold domains that frame the core substrate-binding sites, and four motifs were identified. Furthermore, 25 conserved sites involving these domains were subjected to site-directed mutagenesis, and all mutations resulted in an impact on transport abilities. In particular, as candidates for Na+ -binding sites, D166 and D405 mutations at hairpin discontinuities were detrimental to transport activities and were found to induce pronounced conformational changes using fluorescence resonance energy transfer (FRET) assays. In addition, as observed in the NhaA structure, some charged residues, for example, E64, E65, R454, and R464, are predicted to be involved in the net charge switch of NhaD activation, by collectively form a "pH sensor" at the entrance of the cytoplasmic funnel. Mutations encompassing these residues were detrimental to the transport activity of NhaD or lost the capacity to respond to pH signals and trigger conformational changes for Na+ translocation.
Assuntos
Antiporters , Proteínas de Escherichia coli , Sequência de Aminoácidos , Antiporters/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Prótons , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
The arsenical resistance-3 (ACR3) family constitutes the most common pathway that confers high-level resistance to toxic metalloids in various microorganisms and lower plants. Based on the structural model constructed by AlphaFold2, the Acr3 antiporter from Bacillus subtilis (Acr3Bs ) exhibits a typical NhaA structure fold, with two discontinuous helices of transmembrane (TM) segments, TM4 and TM9, interacting with each other and forming an X-shaped structure. As the structural information available for these important arsenite-efflux pumps is limited, we investigated the evolutionary conservation among 300 homolog sequences and identified three conserved motifs in both the discontinuous helices and TM5. Through site-directed mutagenesis, microscale thermophoresis (MST), and fluorescence resonance energy transfer (FRET) analyses, the identified Motif C in TM9 was found to be a critical element for substrate binding, in which N292 and E295 are involved in substrate coordination, while R118 in TM4 and E322 in TM10 is responsible for structural stabilization. In addition, the highly conserved residues on Motif B of TM5 are potentially key factors in the protonation/deprotonation process. These consensus motifs and residues are essential for metalloid compound translocation of Acr3 antiporters, by framing the core domain and the typical X-shaped of NhaA fold.
Assuntos
Antiporters , Arsenitos , Antiporters/genética , Antiporters/metabolismo , Arsenitos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Estrutura Secundária de ProteínaRESUMO
Nicotine and its major metabolite cotinine are widely used as markers of tobacco smoke abstinence as well as indicators of active smoking levels and the assessment of passive inhalation of tobacco smoke in nonsmokers. Therefore, using an easy-to-prepare sensing platform that can provide a rapid, highly sensitive response for the simultaneous detection of salivary nicotine levels and urinary cotinine levels is especially crucial for helping heavy cigarette smokers quit smoking and protecting public health. Hydrogen-bonded organic frameworks, as a novel class of porous crystalline materials, show immense potential for functional modification and optical sensing. Herein, a new HOF was prepared by a simple solvent evaporation method, and a dual-emitting material Eu(bpy)@HOF-215(1) was obtained by the postsynthetic modification of HOF by lanthanide luminescent complexes, which maintains favorable structural stability and introduces the characteristic emitting of Eu, allowing use as a ratiometric fluorescent sensor for salivary nicotine and urinary cotinine, with a limit of detection of nicotine of 0.045 µM in saliva and a limit of detection of cotinine of 0.591 µM in urine. Furthermore, luminescent inks based on HOF-215 have been fabricated based on the photoresponse variations of 1 to NIC and COT, which enables the multilevel encryption and decryption of information, in a dynamic and recyclable process. This work not only synthesizes a novel blue HOF but also provides a representative successful case of a dual-function platform for simultaneous application to ratiometric sensing and dynamic anticounterfeiting.
Assuntos
Nicotina , Poluição por Fumaça de Tabaco , Nicotina/urina , Cotinina/urina , Poluição por Fumaça de Tabaco/análise , Água , Fumar/metabolismoRESUMO
Carbon-based single atom catalysts (SACs) are attracting extensive attention in the CO2 reduction reaction (CO2RR) due to their maximal atomic utilization, easily regulated active center and high catalytic activity, in which the coordination environment plays a crucial role in the intrinsic catalytic activity. Taking NiN4 as an example, this study reveals that the introduction of different numbers of S atoms into N coordination (Ni-NxS4-x (x = 1-4)) results in outstanding structural stability and catalytic activity. Owing to the additional orbitals around -1.60 eV and abundant Ni dxz, dyz, dx2, and dz2 orbital occupation after S substitution, N,S coordination can effectively facilitate the protonation of adsorbed intermediates and thus accelerate the overall CO2RR. The CO2RR mechanisms for CO and HCOOH generation via two-electron pathways are systematically elucidated on NiN4, NiN3S1 and NiN2S2. NiN2S2 yields HCOOH as the most favorable product with a limiting potential of -0.24 V, surpassing NiN4 (-1.14 V) and NiN3S1 (-0.50 V), which indicates that the different S-atom substitution of NiN4 has considerable influence on the CO2RR performance. This work highlights NiN2S2 as a high-performance CO2RR catalyst to produce HCOOH, and demonstrates that N,S coordination is an effective strategy to regulate the performance of atomically dispersed electrocatalysts.
RESUMO
Electrochemical CO2 reduction reaction (CO2RR) to high-value-added products is one of the most promising strategies for mitigating the greenhouse effect and energy shortage. Two-dimensional (2D) MXene materials are regarded as promising catalysts for electrocatalysis, and the boron-analogs of MXenes, 2D transition metal borides (MBenes), may exhibit superior CO2RR performance owing to their unique electronic properties. Herein, a novel 2D transition metal boride, MoB, is theoretically evaluated as a potential catalyst for the CO2RR by comparing it with traditional Mo2C. MoB shows metallic nature and exhibits excellent electrical conductivity. MoB can effectively activate CO2 with a larger interaction energy of -3.64 eV than that of Mo2C. Both density of states and charge difference density reveal a significant charge transfer from MoB to CO2. MoB shows higher catalytic selectivity due to its inhibited hydrogen evolution reaction and low reaction energy for the CO2RR. At potentials more negative than -0.62 V, the CO2RR on MoB becomes a high-throughput reaction process towards CH4. This work discovered that MoB exhibited comparable CO2RR performance to Mo2C and forecasted MBenes as promising catalysts for electrocatalysis.
RESUMO
BACKGROUND: It is unclear whether Ureaplasma-associated pneumonia and azithromycin treatment affect the risk for bronchopulmonary dysplasia (BPD). METHODS: A retrospective cohort study was performed in very low birth weight (VLBW) infants who tested positive for Ureaplasma within 72 h after birth in a tertiary unit. Chest X-ray (CXR) and laboratory test were performed before and after azithromycin treatment. Multivariate logistic regression analysis was used to identify the independent association between BPD and Ureaplasma-associated pneumonia, as well as BPD and effective azithromycin treatment. RESULTS: A total of 118 infants were included in the current study, of whom 36 developed BPD (defined as supplemental oxygen needed at postmenstrual age 36 weeks or discharge). The rate of BPD was significantly higher in infants with Ureaplasma-associated pneumonia (44.6%) compared to infants with Ureaplasma colonization (17.7%, P = 0.002). After adjusting for confounders, an effective azithromycin treatment was significantly associated with reduced risk of BPD [odd ratio (OR) 0.011; 95% confidence interval (CI): 0.000-0.250), whereas Ureaplasma-associated pneumonia was not significantly associated with BPD (OR 1.835; 95% CI: 0.548-6.147). CONCLUSION: Effective Azithromycin treatment in Ureaplasma positive VLBW infants was associated with a reduced risk of BPD.
Assuntos
Displasia Broncopulmonar , Infecções por Ureaplasma , Recém-Nascido , Humanos , Lactente , Azitromicina/uso terapêutico , Displasia Broncopulmonar/epidemiologia , Recém-Nascido Prematuro , Ureaplasma , Estudos de Coortes , Estudos Retrospectivos , Infecções por Ureaplasma/complicações , Infecções por Ureaplasma/tratamento farmacológicoRESUMO
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in human and animal health care to reduce persistent inflammation, pain and fever because of their anti-inflammatory, analgesic and antipyretic effects. However, the improper discharge and disposal make it becomes a major contaminant in the environment, which poses a big threat to the ecosystem. For this reason, accurate, sensitive, effective, green, and economic techniques are urgently required and have been rapidly developed in recent years. This review summarizes the advancement of sample preparation technologies for NSAIDs involving solid-phase extraction, solid-phase microextraction, liquid-phase microextraction, QuEChERS, and matrix solid-phase dispersion. Meanwhile, we overview and compare analytical technologies for NSAIDs, including liquid chromatography-based methods, gas chromatography-based methods, capillary electrophoresis, and sensors, particularly the development of liquid chromatography-based methods. Furthermore, we focus on their progress and conduct a comparison between their advantages and disadvantages.
Assuntos
Ecossistema , Microextração em Fase Líquida , Animais , Humanos , Anti-Inflamatórios não Esteroides/análise , Cromatografia Líquida , Microextração em Fase Líquida/métodos , Extração em Fase SólidaRESUMO
OBJECTIVES: The NorA antiporter of Staphylococcus aureus belongs to the major facilitator superfamily (MFS) and extrudes various kinds of drugs. With no structure available for this drug efflux pump, the aim of this study was to explore its important structural elements that contribute to substrate binding and drug transport. METHODS: Evolutionary conservative analyses were conducted on different compilations of NorA homologues to identify conservative motifs and residues. Site-directed mutations were constructed to verify the functional changes in NorA efflux capacities and the conformational changes were further measured by fluorescence resonance energy transfer (FRET) and microscale thermophoresis (MST) analysis. RESULTS: Besides Motif-A, Motif-B and Motif-C that were reported previously in MFS proteins, two other motifs, Motif-1 and Motif-2, were identified in NorA. Site-directed mutations of Motif-1 and Motif-2 as well as 11 predicted binding sites all caused remarkable reductions in drug resistance and efflux activity. Among these, mutant F16A/E222A/F303A/D307A showed an altered binding affinity for tetraphenylphosphonium chloride when measured by MST and Motif-1 mutant G114D/A117E/D118G/V119I and Motif-2 mutant Q325E/G326E/A328E/G330E displayed obvious conformational alterations when compared with the wild-type NorA in the FRET signal spectra. CONCLUSIONS: The NorA structure agrees well with the typical structures of MFS proteins, with two newly identified motifs (Motif-1 and Motif-2) that are critical to the structural stability of NorA, and sites F16, E222, F303 and D307 are involved in substrate binding.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
OBJECTIVES: The NorA efflux pump in Staphylococcus aureus mediates resistance to many fluoroquinolone (FQ) antibiotics. Three norA alleles with high sequence similarity are found in various S. aureus strains exhibiting different FQ resistance profiles. This study aimed to elucidate the underlying molecular basis for the varying efflux activity of these three allelic variations. METHODS: The norA genotypes of 20 S. aureus isolates were analysed. Multiple alignments and conservative analyses were conducted to explore the evolutionary variations. After heterologous expression in Escherichia coli, seven mutants were constructed for MIC tests, efflux activity and conformational change measurements. RESULTS: Three NorA alleles were identified that displayed different FQ MICs and varying efflux activity for ethidium bromide, with the NorAII protein showing the strongest activity. A total of 29 single amino acid polymorphisms were identified by conservative analysis within three allelic peptides, with seven sites densely distributed in the 277-297 region. Mutations of these seven residues in NorAII all significantly impaired drug resistance and efflux activity, and three key mutants showed conformational changes in fluorescence resonance energy transfer (FRET) analysis. CONCLUSIONS: Evolutionary variations of the 277-297 region could be a major explanation for the functional difference of three norA alleles and serve as a potential target for the development of novel NorA inhibitors.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos , Staphylococcus aureus , Alelos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
OBJECTIVES: For the stress from fermenters, downstream processing equipment, and wastewater treatment to be alleviated, lowering salt-dependence in the ectoine synthesis process is of great significance in the moderately halotolerant Halomonas hydrothermalis Y2. RESULTS: In H. hydrothermalis Y2, the σ70- and σ38-controlled promoters of ectA are predicted to be involved in the osmotic regulation of ectoine synthesis. By substituting the ectA promoter with a promoter P265 that identified in the outer membrane pore protein E of H. hydrothermalis Y2, the salt dependence of ectoine synthesis was significantly decreased. In the 500-ml flask containing various NaCl contents, the engineered strain (p/Y2/â³ectD/â³doeA) showed a remarkably enhanced ability in ectoine synthesis, especially under lower saline stress. After a 36-h fed-batch fermentation in the 1-l fermenter, p/Y2/â³ectD/â³doeA synthesized 11.5 g ectoine l-1 in the presence of 60 g NaCl-1 l, with a high 0.32 g ectoine l-1 h-1 productivity, a specific productivity of 512.2 mg ectoine per g cell dry weight (CDW)-1, and an excretion ratio of 67 % ectoine. CONCLUSIONS: As no impaired growth was observed in strain p/Y2/â³ectD/â³doeA while ectoine synthesis was increased, this promoter engineering strategy provides a practical protocol for lowering the salt-dependence of ectoine synthesis in this moderately halotolerant strain.
Assuntos
Diamino Aminoácidos/biossíntese , Proteínas de Bactérias/genética , Técnicas de Cultura Celular por Lotes/métodos , Halomonas/crescimento & desenvolvimento , Proteínas da Membrana Bacteriana Externa/genética , Reatores Biológicos/microbiologia , Meios de Cultura/química , Fermentação , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Halomonas/metabolismo , Regiões Promotoras Genéticas , Cloreto de Sódio/químicaRESUMO
BACKGROUND: Lymph node metastasis is a major prognostic sign of colorectal carcinoma and an important indicator for individualized treatment. M2 macrophages play a key role in carcinogenesis and tumor development by enhancing invasiveness and promoting lymph node metastasis. The purpose of this study was to investigate the effect of CD163-positive M2 macrophages on lymph node metastasis in colorectal carcinoma. METHODS: Postoperative lymph node tissues were obtained from 120 patients with colorectal carcinoma who underwent radical surgery in the First Affiliated Hospital of Jinzhou Medical University between December 2019 and May 2020. We detected the expression of the CD163 protein in lymph nodes using immunohistochemistry. Furthermore, the relationships between M2 macrophages identified by expression of CD163 and lymph node metastasis were analyzed using the independent sample t-test and Chi-square test. RESULTS: M2 macrophages were increased in metastatic lymph nodes and non-metastatic lymph nodes adjacent to the cancer. The M2 macrophage count was higher in patients with macro-metastases than in patients with micro-metastases. CONCLUSIONS: The presence of M2 macrophages represents an important indicator for lymph node metastasis in colorectal carcinoma and may be a potential marker for its prediction. Thus, M2 macrophage localization might offer a new target for the comprehensive treatment of colorectal carcinoma.