Estudio computacional de las interacciones moleculares entre el timol y los residuos HIS41 y CYS145 presentes en el sitio activo de la proteasa 3CLpro

La proteína proteasa 3CLpro del SARS-CoV-2 es una enzima crucial para la replicación viral, razón por la cual se convierte en un blanco terapéutico de gran importancia. El timol (2-isopropil-5-me-tilfenol), un compuesto natural que se encuentra en el tomillo (Thymus vulgaris), exhibe potencial actividad antiviral contra la proteasa 3CLpro. En este estudio, usando acoplamiento molecular con AutoDockTools-1.5.6, se evaluaron las energías de interacción molecular entre el timol y los residuos de aminoácidos en el sitio activo de la proteína proteasa 3CLpro. Luego, con la teoría cuántica de Átomos en Moléculas (QTAIM) y la de Interacciones no covalentes (NCI) se analizaron los tipos de interacciones moleculares entre los residuos de aminoácidos identificados y el timol. Los cálculos cuánticos se llevaron con el software Orca-5.0.3, utilizando el método DFT con el funcional M06-2X y el conjunto base aug-cc-pVDZ en fase gaseosa. Los resultados de acoplamiento molecular indican que el timol se une a la proteína 3CL con una energía de interacción igual a -3,784 kcal/mol. El análisis QTAIM indica la presencia de puntos críticos de enlace entre el timol y los residuos HIS41 y CYS145. Además, se observa la formación de un enlace de hidrógeno entre el grupo OH del timol y el residuo CYS145, lo cual es corroborado por los análisis ELF (Electron Localization Function) y NCI (Non Covalent Interactions). Finalmente, el método NCI confirma la presencia de interacciones de Van der Waals con el residuo HIS41. Los resultados sugieren que el mecanismo de inhibición de la actividad de la proteína 3CLpro es controlado por interacciones moleculares tipo puente de hidrógeno e interacciones débiles.

The protease 3CLpro of the SARS-CoV-2 is a crucial enzyme for viral replication, becoming a highly important therapeutic target. Thymol (2-isopropyl-5-methyl-phenol), a naturally occurring compound found in thyme, exhibits potential antiviral activity against the 3CLpro protease. In this study, using molecular docking with AutoDockTools-1.5.6, the molecular interaction energies between thymol and amino acid residues in the active site of the protein protease 3CLpro were evaluated. Then, with the Atoms in Molecules (QTAIM) and Non-covalent Interactions (NCI) theories, the types of molecular interactions between identified amino acid residues and thymol were analyzed. Quantum calculations were carried out with the Orca-5.0.3 software using the DFT method with the M06-2X functional and the aug-cc-pVDZ basis set in the gas phase. The molecular docking results indicate that thymol is linked to the 3CL protein with an interaction energy equal to -3.784 kcal/mol. QTAIM analysis indicates the presence of critical binding sites between thymol and residues HIS41 and CYS145. In addition, the formation of a hydrogen bond between the OH group of thymol and the CYS145 residue is observed, which is corroborated by the ELF and NCI analyses. Finally, the NCI method confirms the presence of Van der Waals interactions with the HIS41 residue. The results suggest that the mechanism of inhibition of the activity of the 3CLpro protein is controlled by molecular interactions such as hydrogen bonding and weak interactions.

A protease 3CLpro do SARS-CoV-2 é uma enzima crucial para a replicação viral, tornando-se um alvo terapêutico de grande importÅncia. O timol (2-isopropil-5-me-tilfenol), um composto natural encontrado no tomilho, exibe potencial atividade antiviral contra a protease 3CLpro. Neste estudo, utilizando o docking molecular com o AutoDockTools-1.5.6, foram avaliadas as energias de interação molecular entre o timol e os residuos de aminoácidos no sítio ativo da proteína protease 3CLpro. Em seguida, com a teoria quantica de atomos em moleculas (QTAIM) e da interacões no-covalentes (NCI), foram analisados os tipos de interações moleculares entre os resíduos de aminoácidos identificados e o timol. Os cálculos quÅnticos foram realizados com o software Orca-5.0.3 usando o método DFT com o funcional M06-2X e a base aug-cc-pVDZ definida na fase gasosa. Os resultados do docking molecular indicam que o ti-mol está ligado à proteína 3CL com uma energia de interação igual a -3.784 kcal/ mol. A análise QTAIM indica a presença de sítios de ligação críticos entre o timol e os resíduos HIS41 e CYS145. Além disso, observa-se a formação de uma ponte de hidrogênio entre o grupo OH do timol e o resíduo CYS145, o que é corroborado pelas análises ELF e NCI. Finalmente, o método NCI confirma a presença das interações de Van der Waals com o resíduo HIS41. Os resultados sugerem que o mecanismo de inibição da atividade da proteína 3CLpro é controlado por interações moleculares como ligações de hidrogênio e interações fracas.

Neuroprotective effect and mechanism of Zuogui Jiangtang Jieyu Formula on diabetes mellitus complicated with depression model rats based on CX3CL1-CX3CR1 axis / 中国中药杂志

Based on the CX3C chemokine ligand 1(CX3CL1)-CX3C chemokine receptor 1(CX3CR1) axis, this study explored the potential mechanism by which Zuogui Jiangtang Jieyu Formula(ZGJTJY) improved neuroinflammation and enhanced neuroprotective effect in a rat model of diabetes mellitus complicated with depression(DD). The DD rat model was established by feeding a high-fat diet combined with streptozotocin(STZ) intraperitoneal injection for four weeks and chronic unpredictable mild stress(CUMS) combined with isolated cage rearing for five weeks. The rats were divided into a control group, a model group, a positive control group, an inhibitor group, and a ZGJTJY group. The open field test and forced swimming test were used to assess the depression-like behaviors of the rats. Enzyme-linked immunosorbent assay(ELISA) was performed to measure the expression levels of the pro-inflammatory cytokines interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) in plasma. Immunofluorescence staining was used to detect the expression of ionized calcium-binding adapter molecule 1(Iba1), postsynaptic density protein-95(PSD95), and synapsin-1(SYN1) in the hippocampus. Hematoxylin-eosin(HE) staining, Nissl staining, and TdT-mediated dUTP nick end labeling(TUNEL) fluorescence staining were performed to assess hippocampal neuronal damage. Western blot was used to measure the expression levels of CX3CL1, CX3CR1, A2A adenosine receptor(A2AR), glutamate receptor 2A(NR2A), glutamate receptor 2B(NR2B), and brain-derived neurotrophic factor(BDNF) in the hippocampus. Compared with the model group, the ZGJTJY group showed improved depression-like behaviors in DD rats, enhanced neuroprotective effect, increased expression of PSD95, SYN1, and BDNF(P<0.01), and decreased expression of Iba1, IL-1β, and TNF-α(P<0.01), as well as the expression of CX3CL1, CX3CR1, A2AR, NR2A, and NR2B(P<0.01). These results suggest that ZGJTJY may exert its neuroprotective effect by inhibiting the CX3CL1-CX3CR1 axis and activation of hippocampal microglia, thereby improving neuroinflammation and abnormal activation of N-methyl-D-aspartate receptor(NMDAR) subunits, and ultimately enhancing the expression of synaptic-related proteins PSD95, SYN1, and BDNF in the hippocampus.

Assessment Of Gingival Phenotype At The Aesthetic Zone In A Malay Population Using Probe Test

@#Introduction: The gingival phenotype (GP) of teeth at the aesthetic zone often influences dental rehabilitation plans and treatment outcomes. This study aimed to assess the prevalence of GP in the Malay population in relation to gender and age. Methods: The GP of 100 patients were determined using the Probe test method. Other clinical parameters were assessed include crown width/crown length (CW/CL) ratio, tooth morphology and width of keratinised tissue. Periodontal parameters were assessed by two calibrated examiners. Data were analysed using descriptive statistics, one-way ANOVA and Kruskal-Wallis test. Results: A higher prevalence of thick GP was found at the maxilla for both genders, whereas a thin phenotype was observed at the mandible. At maxilla, both thick and thin GP were found in all age groups, while the mandible showed a higher prevalence of thin GP. Significant differences in GP were found between males and females for mandibular and maxillary anterior teeth and the mandibular lateral incisor (p<0.05), while no significant difference was found for other parameters assessed; age group, CW/CL, tooth morphology and WKT. Conclusion: Thicker GP is more prevalent in male population and at maxillary anterior. Mandibular anterior GP presented commonly with a thin GP regardless of gender or age-group.

Control of sodium and potassium homeostasis by renal distal convoluted tubules

Distal convoluted tubules (DCT), which contain the Na-Cl cotransporter (NCC) inhibited by thiazide diuretics, undergo complex modulation to preserve Na+ and K+ homeostasis. The lysine kinases 1 and 4 (WNK1 and WNK4), identified as hyperactive in the hereditary disease pseudohypoaldosteronism type 2, are responsible for activation of NCC and consequent hypokalemia and hypertension. WNK4, highly expressed in DCT, activates the SPAK/OSR1 kinases, which phosphorylate NCC and other regulatory proteins and transporters in the distal nephron. WNK4 works as a chloride sensor through a Cl- binding site, which acts as an on/off switch at this kinase in response to changes of basolateral membrane electrical potential, the driving force of cellular Cl- efflux. High intracellular Cl- in hyperkalemia decreases NCC phosphorylation and low intracellular Cl- in hypokalemia increases NCC phosphorylation and activity, which makes plasma K+ concentration a central modulator of NCC and of K+ secretion. The WNK4 phosphorylation by cSrc or SGK1, activated by angiotensin II or aldosterone, respectively, is another relevant mechanism of NCC, ENaC, and ROMK modulation in states such as volume reduction, hyperkalemia, and hypokalemia. Loss of NCC function induces upregulation of electroneutral NaCl reabsorption by type B intercalated cells through the combined activity of pendrin and NDCBE, as demonstrated in double knockout mice (KO) animal models, Ncc/pendrin or Ncc/NDCBE. The analysis of ks-Nedd-4-2 KO animal models introduced the modulation of NEDD4-2 by intracellular Mg2+ activity as an important regulator of NCC, explaining the thiazide-induced persistent hypokalemia.

El mejillón Semimytilus patagonicus (Mytilida: Mytilidae) y el gusano Pseudonereis gallapagensis (Phyllodocida: Nereididae) como herramientas ecotoxicológica para la contaminación por detergentes en ambientes marinos / The mussel Semimytilus patagonicus (Mytilida: Mytilidae) and the worm Pseudonereis gallapagensis (Phyllodocida: Nereididae) as ecotoxicological tools for detergent pollution in marine environments

Introduction: The bivalve Semimytilus patagonicus is a potentially useful bioindicator because of its feeding mechanism, and the worm Pseudonereis gallapagensis is also interesting as a bioindicator because it is benthonic, abundant, and a food source for the squid Doryteuthis gahi. However, their sensitivity to contaminants has not been sufficiently studied. Objective: To test the usefulness of the mussel Semimytilus patagonicus and the polychaete Pseudonereis gallapagensis as ecotoxicological tools for detergents in the marine environment. Methods: We used 120 individuals of S. patagonicus from Miraflores and 120 of P. gallapagensis from Barranco (both near the city of Lima, Peru). For the bioassays, we used two anionic detergents (active ingredient, ai, Sodium Dodecylbenzene Sulfonate). For S. patagonicus, with an average valve length of 32.3 ± 6.4 mm, we tested "Double power Ariel®" (90 %) at concentrations of 17.5, 35, 70 and 140 mg ai l-1, evaluated after 48 and 72 h of exposure; and for P. gallapagensis, with a total body length of 20.4 ± 8.8 mm, we tested "Caricia®" at 62.5, 125, 250, 500 and 1 000 mg of ai l-1 at 24, 48 and 72 h of exposure. Results: The LC50 values (Mean Lethal Concentration) were 34.95 mg ia l-1 for S. patagonicus and 102.48 mg ia l-1 for P. gallapagensis at 72 h of exposure. The detergents were toxic for S. patagonicus and slightly toxic for P. gallapagensis. The risk classification for S. patagonicus is "harmful" and for P. gallapagensis "not classifiable". Conclusions: These two bioindicators allow evaluating the acute toxicity of SDBS-based commercial detergents in the marine aquatic environment.

Introducción: El bivalvo Semimytilus patagonicus es un bioindicador potencialmente útil por su mecanismo de alimentación, y el gusano Pseudonereis gallapagensis también es interesante como bioindicador por ser bentónico, abundante y fuente de alimento para el calamar Doryteuthis gahi. Sin embargo, su sensibilidad a los contaminantes no ha sido suficientemente estudiada. Objetivo: Probar la utilidad del mejillón S. patagonicus y el poliqueto P. gallapagensis como herramientas ecotoxicológicas para detergentes en el medio marino. Métodos: Se utilizaron 120 individuos de S. patagonicus de Miraflores y 120 de P. gallapagensis de Barranco (ambos cerca de la ciudad de Lima, Perú). Para los bioensayos se utilizaron dos detergentes aniónicos (ingrediente activo, ia, dodecilbenceno sulfonato de sodio). Para S. patagonicus, con una longitud valver promedio de 32.3 ± 6.4 mm, probamos Ariel Doble Poder® (90 %) a concentraciones de 17.5, 35, 70 y 140 mg·ia·l-1, evaluadas a las 48 y 72 h de exposición; y para P. gallapagensis, con una longitud corporal total de 20.4 ± 8.8 mm, probamos Caricia® a 62.5, 125, 250, 500 y 1 000 mg·ia·l-1 a las 24, 48 y 72 h de exposición. Resultados: Los valores de CL50 (Concentración Letal Media) fueron de 34.95 mg·ia·l-1 para S. patagonicus y 102.48 mg·ia·l-1 para P. gallapagensis a las 72 h de exposición. Los detergentes fueron tóxicos para S. patagonicus y levemente tóxicos para P. gallapagensis. La clasificación de riesgo para S. patagonicus es "nocivo" y para P. gallapagensis "no clasificable". Conclusiones: Estos dos bioindicadores permiten evaluar la toxicidad aguda del detergente comercial a base de SDBS en el ambiente acuático marino.

Multi-level scoring approach to discover multi-targeting potency of medicinal plant phytochemicals against protein targets in SARS-CoV-2 and human ACE-2 receptor

SARS-CoV-2 pandemic has become a major threat to human healthcare and world economy. Due to the rapid spreading and deadly nature of infection, we are in a situation to develop quick therapeutics to combat SARS-CoV-2. In this study, we have adopted a multi-level scoring approach to identify multi-targeting potency of bioactive compounds in selected medicinal plants and compared its efficacy with two reference drugs, Nafamostat and Acalabrutinib which are under clinical trials to treat SARS-CoV-2. In particular, we employ molecular docking and implicit solvent free energy calculations (as implemented in the Molecular Mechanics -Generalized Born Surface Area approach) and QM fragmentation approach for validating the potency of bioactive compounds from the selected medicinal plants against four di?erent viral targets and one human receptor (Angiotensin-converting enzyme 2 -ACE-2) which facilitates the SARS-CoV-2entry into the cell. The protein targets considered for the study are viral 3CL main protease (3CLpro), papain-like protease (PLpro), RNA dependent RNA polymerase (RdRp), and viral spike protein-human hACE-2 complex (Spike:hACE2)including human protein target (hACE-2). Herein, thereliable multi-level scoring approach was used to validate the mechanism behind the multi-targeting potency of selected phytochemicals from medicinal plants. The present study evidenced that the phytochemicals Chebulagic acid, Stigmosterol, Repandusinic acid and Geranin exhibited efficient inhibitory activity against PLpro while Chebulagic acid was highly active against 3CLpro. Chebulagic acid andGeranin also showed excellent target specific activity against RdRp.Luteolin, Quercetin, Chrysoeriol and Repandusinic acid inhibited the interaction of viral spike protein with human ACE-2 receptor. Moreover Piperlonguminine and Piperine displayed significant inhibitory activity against human ACE-2 receptor. Therefore, the identified compounds namely Chebulagic acid, Geranin and Repandusinic acid can serve as potent multi-targeting phytomedicine for treating COVID-19

Development Of Halosulphate Based Phosphors And It's Applications

Synthesis and characterization of halosulphate-based phosphors is important for thermoluminescence dosimetry (TLD), radiophotoluminescence dosimetry (RPL) and scintillator materials. The enhancement of luminescence output in halosulphate-based phosphors and it may be useful for lamp, solid-state lamp and radiation. Dosimetry by activator as well as sensitizer are well known properties. The combustion technique is not applicable for the synthesis of TLD phosphors due to very fine particles, which show less TL intensity, while sol-gel, solid-state diffusion, melt method and precipitation methods are applicable for TLD phosphors. Two halosulphates namely Na21( SO4 ) 7 F6 Cl and 2K3Ca2(SO4)3F were prepared and doped with Dy and Tm for different concentration .Halosulphate , Na21( SO4 ) 7 F6 Cl was prepared by wet chemical method and Halosulphate , 2K3Ca2(SO4)3F was prepared by solid state diffusion method . The characterization was done by X - ray diffraction ( XRD ) , Thermo luminescence (TL) was also studied . For Dy doped Na21( SO4) 7 F6 Cl , The peak was observed at 1200 C and shoulder at 1750C for 0.2 % molar concentration of Dy. and for 2K3Ca2(SO4)3F doped with Tm the shoulder peak was observed at 240 0 C and at 150 0C for 0.7 % molar concentration of Tm.

Apigenin Induces Apoptosis of Human Colorectal Cancer CL187 Cells via PI3K/Akt and MAPK Signaling Pathways / 中国实验方剂学杂志

ObjectiveTo study the effect of apigenin on the proliferation and apoptosis of human colon cancer CL187 cells and the underlying mechanisms. MethodHuman colorectal cancer CL187 cells were treated with different concentrations of apigenin （0， 30， 45， 60 mg·L-1） according to the results of the preliminary experiment. The proliferation of CL187 cells was detected by methyl thiazolyl tetrazolium （MTT） and colony formation assays， and the apoptosis was observed via Hoechst 33258 staining. Real-time fluorescence quantitative PCR was conducted to determine the mRNA levels of cysteine protease-3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in the CL187 cells treated with apigenin. Western blot was employed to measure the protein levels of Caspase-3， Bcl-2， and Bax associated with apoptosis， protein kinase B （Akt） and phosphorylated Akt （p-Akt） in phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） pathway， and extracellular signal-regulated kinases 1/2 （ERK1/2）， p-ERK1/2， c-Jun N-terminal kinase （JNK）， p-JNK， p38 mitogen-activated protein kinase （MAPK）， and p-p38 MAPK protein in MAPK pathway. ResultCompared with the blank group， the apigenin groups had low cell survival rates and high inhibition rates on cell proliferation （P<0.01）. Apigenin decreased the cell clone number and clone formation rate， and increased the inhibition rate on clone formation （P<0.01）. After CL187 cells were treated with different concentrations of apigenin for 48 h， typical apoptosis characteristics such as nuclear pyknosis， chromatin condensation， and enhanced fluorescence reaction were observed. Compared with blank group， 45， 60 mg·L-1 apigenin treatments down-regulated the mRNA level of anti-apoptotic gene Bcl-2 （P<0.01） and all the apigenin treatments up-regulated those of the pro-apoptotic genes Bax and Caspase-3 （P<0.05， P<0.01）. Similarly， apigenin treatments down-regulated the protein level of Bcl-2 （P<0.05， P<0.01） and up-regulated those of Caspase-3 （P<0.05， P<0.01） and Bax （P<0.01， 45， 60 mg·L-1）. The blank group had higher protein level of Akt than the 60 mg·L-1 apigenin group （P<0.01）， higher protein levels of p-Akt， ERK1/2， and p-ERK1/2 than the 45， 60 mg·L-1 apigenin groups （P<0.01）， and higher protein levels of JNK and p-JNK than the apigenin groups （P<0.05， P<0.01）. Compared with blank group， 60 mg·L-1 apigenin up-regulated the protein level of p38 MAPK （P<0.05）， and all the apigenin groups up-regulated that of p-p38 MAPK （P<0.01）. Furthermore， apigenin lowered the p-Akt/Akt ratio （P<0.05， P<0.01） and p-ERK1/2/ERK1/2 ratio （P<0.01）， while it increased the p-JNK/JNK ratio （45， 60 mg·L-1； P<0.05， P<0.01） and p-p38 MAPK/p38 MAPK ratio （P<0.05， P<0.01）. ConclusionApigenin can inhibit the proliferation and promote the apoptosis of CL187 cells by inhibiting the PI3K/Akt signaling pathway and regulating the expression of proteins in the MAPK signaling pathway.

Apigenin Induces Apoptosis of Human Colorectal Cancer CL187 Cells via PI3K/Akt and MAPK Signaling Pathways / 中国实验方剂学杂志

ObjectiveTo study the effect of apigenin on the proliferation and apoptosis of human colon cancer CL187 cells and the underlying mechanisms. MethodHuman colorectal cancer CL187 cells were treated with different concentrations of apigenin （0， 30， 45， 60 mg·L-1） according to the results of the preliminary experiment. The proliferation of CL187 cells was detected by methyl thiazolyl tetrazolium （MTT） and colony formation assays， and the apoptosis was observed via Hoechst 33258 staining. Real-time fluorescence quantitative PCR was conducted to determine the mRNA levels of cysteine protease-3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in the CL187 cells treated with apigenin. Western blot was employed to measure the protein levels of Caspase-3， Bcl-2， and Bax associated with apoptosis， protein kinase B （Akt） and phosphorylated Akt （p-Akt） in phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） pathway， and extracellular signal-regulated kinases 1/2 （ERK1/2）， p-ERK1/2， c-Jun N-terminal kinase （JNK）， p-JNK， p38 mitogen-activated protein kinase （MAPK）， and p-p38 MAPK protein in MAPK pathway. ResultCompared with the blank group， the apigenin groups had low cell survival rates and high inhibition rates on cell proliferation （P<0.01）. Apigenin decreased the cell clone number and clone formation rate， and increased the inhibition rate on clone formation （P<0.01）. After CL187 cells were treated with different concentrations of apigenin for 48 h， typical apoptosis characteristics such as nuclear pyknosis， chromatin condensation， and enhanced fluorescence reaction were observed. Compared with blank group， 45， 60 mg·L-1 apigenin treatments down-regulated the mRNA level of anti-apoptotic gene Bcl-2 （P<0.01） and all the apigenin treatments up-regulated those of the pro-apoptotic genes Bax and Caspase-3 （P<0.05， P<0.01）. Similarly， apigenin treatments down-regulated the protein level of Bcl-2 （P<0.05， P<0.01） and up-regulated those of Caspase-3 （P<0.05， P<0.01） and Bax （P<0.01， 45， 60 mg·L-1）. The blank group had higher protein level of Akt than the 60 mg·L-1 apigenin group （P<0.01）， higher protein levels of p-Akt， ERK1/2， and p-ERK1/2 than the 45， 60 mg·L-1 apigenin groups （P<0.01）， and higher protein levels of JNK and p-JNK than the apigenin groups （P<0.05， P<0.01）. Compared with blank group， 60 mg·L-1 apigenin up-regulated the protein level of p38 MAPK （P<0.05）， and all the apigenin groups up-regulated that of p-p38 MAPK （P<0.01）. Furthermore， apigenin lowered the p-Akt/Akt ratio （P<0.05， P<0.01） and p-ERK1/2/ERK1/2 ratio （P<0.01）， while it increased the p-JNK/JNK ratio （45， 60 mg·L-1； P<0.05， P<0.01） and p-p38 MAPK/p38 MAPK ratio （P<0.05， P<0.01）. ConclusionApigenin can inhibit the proliferation and promote the apoptosis of CL187 cells by inhibiting the PI3K/Akt signaling pathway and regulating the expression of proteins in the MAPK signaling pathway.

Screening of the ubiquitin-proteasome system activators for anti-Alzheimer's disease by the high-content fluorescence imaging system / 中国天然药物

Ubiquitin-proteasome system (UPS) plays an important role in neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important. In this study, we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD. At first, stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells, together with G418 screening. The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1. By employing the high-content fluorescence imaging system, together with stable YFP-CL1 HT22 cells, the UPS activators were successfully screened from our established TCM library. The representative images were captured and analyzed, and quantification of the YFP fluorescence intensity was performed by flow cytometry. Then, the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP (APP), pRK5-EGFP-Tau P301L (Tau P301L), or pRK5-EGFP-Tau (Tau) transiently transfected HT22 cells using fluorescence imaging, flow cytometry, and Western blot. In conclusion, our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the high-throughput screening of the UPS activators. Three compounds, namely salvianolic acid A (SAA), salvianolic acid B (SAB), and ellagic acid (EA), were identified to significantly decrease YFP fluorescence intensity, which suggested that these three compounds are UPS activators. The identified UPS activators were demonstrated to clear AD-related proteins, including APP, Tau, and Tau P301L. Therefore, these findings provide a novel insight into the discovery and development of anti-AD drugs.

TOXICIDAD AGUDA DEL HERBICIDA PARAQUAT EN Oreochromis niloticus (CICHLIDAE) Y Macrobrachium olfersii (PALAEMONIDAE) / Paraquat's herbicide acute toxicity in Oreochromis niloticus (Cichlidae) and Macrobrachium olfersii (Palaemonidae)

RESUMEN El Paraquat es un herbicida utilizado en la actividad agropecuaria para controlar hierbas, su modo de acción es por medio de contacto y no selectivo. Debido a su alta solubilidad en agua y baja volatilidad representa un riesgo potencial para organismos acuáticos, principalmente los que son cultivados con aguas superficiales que reciben impacto de la actividad agrícola. La tilapia Oreochromis niloticus y el langostino Macrobrachium olfersii son organismos de importancia comercial para la industria acuícola del estado de Veracruz, México. El objetivo de este estudio fue determinar la Concentración Letal Media (CL50) del herbicida Dasurquat® (ingrediente activo Paraquat) a través de un bioensayo de toxicidad aguda (96 horas). Se utilizaron como especímenes de prueba a juveniles de tilapia O. niloticus (peso promedio = 10 mg, longitud total= 8,75 mm) y otro el ensayo fue con poslarvas de langostino M. olfersii (peso promedio = 5 mg, longitud total= 5,72 mm). Se emplearon cinco concentraciones (5, 10, 20, 40 y 80 μL-1 para el bioensayo con juveniles de tilapia; para el bioensayo con poslarvas de langostino las concentraciones fueron 0,1, 0,2, 0,5, 0,7 y 1 μL-1). El diseño experimental consideró un control negativo, con dos repeticiones y dos réplicas por cada tratamiento. El análisis de datos se realizó con el método Probit para determinar la CL50 a 96 horas, se obtuvo un valor para juveniles de O. niloticus de 17,49 μL-1 con intervalo de confianza (95 %) con límite inferior de 13,75 μL-1 y límite superior 22,25 μL-1, para las larvas de M. olfersii se obtuvo un valor de 0,31 μL-1 con intervalo de confianza (95 %) con límite inferior de 0,26 μL-1 y límite superior 0,35 μL-1. El análisis de varianza demostró que no existió diferencia estadística significativa (p > 0.05) entre las réplicas de los tratamientos. Se concluye que es necesario continuar con estudios para evaluar su toxicidad en organismos acuáticos debido al amplio uso de este herbicida en la actividad agropecuaria, y determinar su riesgo para otras actividades productivas además de la acuicola.

ABSTRACT Paraquat is an herbicide used in the agricultural industry for weed and undergrowth control, its mode of action is by contact and nonselective. Due to its high-water solubility and low volatility, it represents a potential risk for non-target organisms. Oreochromis niloticus tilapia and the prawn Macrobrachium olfersii are important commercial species for Veracruz's aquaculture industry. The objective of this study was to determine the Mean Lethal Concentration (LC50) of the herbicide Dasurquat® (active ingredient Paraquat) through an acute toxicity bioassay (96 hours). Juvenile O. niloticus (average weight = 10 mg, total length = 8.75 mm) and juvenile M. olfersii postlarvae (average weight = 5 mg, total length = 5.72mm) were used as test specimens. Five herbicide concentrations (5, 10, 20, 40 and 80 μL-1 for tilapia and 0.1, 0.2, 0.5, 0.7 y 1 μL-1 for prawn) were applied. The experimental design considered a negative control, with two replications and two replicates for each treatment. Probit analysis determined that the LC50 at 96 hours for O. niloticus was 17.49 μL-1, with a lower 95 % confidence limit of 13.75 μL-1 and an upper limit of 22.25 μL-1, whereas for M. olfersii the LC50 at 96 hours was 0.31 μL-1, with a lower 95 % confidence limit of 0.26 μL-1 and an upper limit of 0.35 μL-1. The analysis of variance showed that there was no significant statistical difference (p > 0.05) between the replicates of the treatments. It is concluded that it is necessary to continue evaluating its toxicity in aquatic organisms due to the wide use of this herbicide in Veracruz's agricultural activity, to determine its potential risk to other activities.

Overexpression of a fusion protein of 4-coumaroyl-CoA ligase and polyketide synthase for raspberry ketone production in Chlamydomonas reinhardtii / 生物工程学报

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 μg/g (fresh weight) and 5.9 μg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2-4 μg/g).

Effect of CX3CL1/CX3CR1 in cerebral ischemia and its mechanisms / 国际脑血管病杂志

CX3CL1, also known as Fractalkine, is the only member of chemokines CX3C subclass. It plays an important role in a variety of central nervous system diseases and ischemic cerebrovascular diseases by binding to its specific receptor CX3CR1. In recent years, a large number of studies have investigated the specific role and related molecular mechanism of CX3CL1/CX3CR1. This article reviews the effect and molecular mechanism of CX3CL1/CX3CR1 in ischemic cerebrovascular disease, aiming to expand the understanding of the mechanism of CX3CL1/CX3CR1, and provide new ideas and intervention targets for the prevention, diagnosis and treatment of ischemic cerebrovascular disease.

Research advances of chemokine receptor CX3CR1 and microglia in retinal degenerative diseases / 国际眼科杂志(Guoji Yanke Zazhi)

@#Retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration are the main clinical blinding eye diseases with complex etiology and irreversible damage to vision. CX3CR1 is a specific receptor of the chemokine CX3CL1. Both of them participate in various physiological functions and pathological changes of the whole body through regulating the immune system of the body. In recent years, studies have pointed out that CX3CR1 regulates the activity and function of retinal microglia, which play an important role in the process of retinal degenerative diseases. In this paper, the structure and function of the chemokine receptor CX3CR1 and the role of microglia in retinal degenerative diseases were reviewed, so as to provide ideas and directions for future research and treatment of such diseases.

Potential active compounds of Liupao tea for prevention and treatment of COVID-19 based on network pharmacology and molecular docking / 中国药科大学学报

@#To investigate the material basis and mechanism of Liupao tea on preventing COVID-19 by network pharmacology and molecular docking.The active ingredients and targets of Liupao tea were searched through the literature and the TCMSP databases and the network between the two was built by Cytoscape 3.7.1.Then using GenCards platform to predict the disease targets,mapping the common targets between Liupao tea and disease.The common targets were imported into the STRING database for exploring the protein-protein interaction.Core targets were enriched by gene ontology (GO) enrichment analysis and KEGG (kyoto encyclopedia of genes and genomes) pathway enrichment analysis using DAVID database etc..Finally,the screened active components were docked with the receptor protein SARS-CoV-2 3CL hydrolase (Mpro).Six active ingredients of Liupao tea were screened,such as (-)-epigallocatechin gallate (EGCG),(+)-catechin,(-)-catechin gallate,α-spinasterol,pelargonidin chloride and squalene,and 156 targets were identified.Among them,there were 112 common targets and 38 core targets with COVID-19.GO enrichment analysis (P<0.01) involved lipopolysaccharide,cell response to hypoxia,etc..And the KEGG pathway enrichment analysis (P<0.01)was conducted to obtain the HIF-1,IL-17,T cell receptor and other signaling pathways associated with COVID-19.The results of molecular docking showed that the active ingredients of Liupao tea were well bound to the receptor protein Mpro.The active ingredients of Liupao tea may control HIF-1,IL-17,T cell receptors signaling pathways by binding Mpro hydrolase and acting on inflammation and immune related targets such as MAPK1,TNF to prevent COVID-19.The EGCG of Mpro activity was determined ,and the IC50 was 3.4 μmol/L,which confirmed that EGCG was a certain inhibition effect on Mpro.

Virtual screening of small molecular inhibitors of SARS-CoV-2 3CL hydrolase based on high-throughput molecular docking and prediction of Chinese materia medica and its compound against COVID-19 / 中草药

Objective: Based on the systematic pharmacological database of traditional Chinese medicine (TCM) and the analysis platform TCMSP, the computer virtual screening technique was used to screen the small molecule inhibitors of SARS-CoV-2 3CL hydrolase from Chinese materia medica (CMM), and speculate the potential anti-COVID-19 novel coronavirus pneumonia TCMs and its compounds. Methods: SARS-CoV-2 3CL hydrolase protein was targeted in this study. Autodock Vina software and Python script were used to realize high-throughput molecular docking. Combined with “ADME-Lipinski” rules, the re-screening was carried out to optimize the active ingredients and speculate the key TCMs and compound prescriptions. Based on the perspective of network pharmacology, a component-target-pathway network was constructed to infer the mechanism of action of core drug pairs. Results: Taking the reference ligand as positive control, 66 natural micromolecule compounds with good pharmacokinetic properties were obtained. Twelve single TCMs, two Chinese medicine pairs of Glycyrrhizae Radix et Rhizoma-Mori Cortex and Lonicerae Japonicae Flos-Forsythiae Fructus, and 12 TCM prescriptions including Sangju Drink and modified Sangju Drink and Yinqiao Powder were selected as candidate schemes to fight against novel coronavirus pneumonia. Conclusion: This study is based on high-throughput molecular docking technology to virtually screen small molecule inhibitors of SARS-CoV-2 3CL hydrolase of CMM and Chinese medicines, innovatively analyze the potential molecular mechanism in combination with network pharmacology, and provide scientific guidance and theoretical basis for TCM to resist novel coronavirus pneumonia.

Exploring potential effect of Shengjiang San on SARS-CoV-2 based on network pharmacology and molecular docking / 中草药

Objective: To explore the potential effect of Shengjiang San for inhibiting SARS-CoV-2. Methods: The target genes of Beauveria bassiana, Cryptotympana pustulata, Curcuma longa, Rheum officinale in Shengjiang San were screened out through the database analysis of Encyclopedia of Traditional Chinese Medicine (ETCM), and traditional Chinese medicine system pharmacology platform (TCMSP), Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) and Collective Molecular Activities of Useful Plants (CMAUP). GeneCards database was used to obtain target genes of antivirus. The intersection method was used to obtain the target genes related to the antiviral effect of Shengjiang San. Cytoscape 3.7.2 software was applied for the construction of prescription-CMM-targets (genes) networks. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and gene ontology (GO) functional enrichment analysis were performed by R language to predict the potential mechanism of Shengjiang San against the virus. TCMSP, CNKI and PubChem databases were used to retrieve the chemical components of B. bassiana, C. pustulata, C. longa and R. officinale in Shengjiang San. AutoDock Vina 1.1.2 was used for molecular docking to study the interactions of each chemical component with SARS-CoV-2 3CL hydrolase or angiotensin converting enzyme II (ACE2). Results: Shengjiang San could play an antiviral role through the corresponding 663 target genes. Top ten pathways were related to antivirus (P < 0.01) in the KEGG pathway enrichment screening, including influenza A, etc. The affinity values of a total of 133 compounds in Shengjiang San were < -29.3 kJ/mol for molecular docking with SARS-CoV-2 3CL hydrolase. The affinity values of 145 compounds for molecular docking with ACE2 were < -29.3 kJ/mol. Conclusion: Shengjiang San could regulate multiple signaling pathways to inhibit virus, and have a potential inhibiting effect on SARS-Cov-2.

Research on active compounds of Maxingyigan Decoction for treatment of coronavirus disease 2019 based on network pharmacology and molecular docking / 中草药

Objective: To explore the active compounds of Maxingyigan Decoction for the treatment of coronavirus disease 2019 (COVID-19). Methods: The chemical constituents and action targets of Ephedra sinica, Armeniacae Semen Amarum, Coicis Semen, and Glycyrrhizae Radix et Rhizoma in Maxingyigan Decoction were retrieved from TCMSP. The database of UniProt and GeneCards were used to query the target genes that corresponding to the active compounds, and then a compound-target (gene) network was constructed by Cytoscape 3.6.1. GO functional enrichment analysis and KEGG enrichment analysis were performed through WebGestalt database to predict its mechanism of action. The main active ingredients were docked with SARS-CoV-2 3CL hydrolase and angiotensin converting enzyme II (ACE2). Results: The compound-target network contained 126 compounds and 266 corresponding targets. The key targets genes included PTGS2, ESR1, PCP4, PPARG, HSP90AA1, NCOA2, etc. GO function enrichment analysis found that 522 GO items were affected by Maxingyigan Decoction, including 12 biological process items, 20 cell composition items, and 17 molecular function items. KEGG enrichment analysis showed that 168 signal pathways were enriched, involving interferon-γ signaling pathway, MAP kinase cascade, T cell activation, chemokines and cytokine signaling pathway-mediated inflammation pathways, etc. The molecular docking results showed that core compounds such as luteolin and quercetin had similar affinity with the recommended drugs used to treat COVID-19. Conclusion: The active compounds in Maxingyigan Decoction may have a therapeutic effect on COVID-19 through binding with 3CL hydrolase and ACE2 to act on targets such as PTGS2, ESR1, PCP4, PPARG, HSP90AA1 and NCOA2 so as to regulate multiple signal pathways.

Potential SARS-CoV-2 3CL protease inhibitors selection from TCMSP platform by molecular docking approach / 中草药

Objective: To identify potential SARS-CoV-2 3CL protease inhibitors from the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) by molecular docking approach. Methods: To alternate extensive compounds experimental screening processes, a Computer-Aided Drug Design (CADD) based molecular docking technology was performed to explore existing drug repurposing possibilities. Molecular docking model with Schrodinger suit 2018 was used to evaluate the binding abilities between TCMSP 13 143 compounds and SARS-CoV-2 3CL protease receptor-binding domain (PBD ID 6LU7), which involving in mediating viral replication and transcription functions. According to the constructed docking system, potential compounds were screened according to docking score, oral bioavailability (OB), and drug-likeness (DL). At last, a compounds-herb-target organ-function network was constructed. Results: Compared with 6LU7 original ligand docking score (-7.734), a total of 498 compounds were identified with lower docking score against 6LU7 targets. These compounds were further reduced to 60 high-priority compounds, based on OB (more than 30) and DL (more than 0.18). Meanwhile, these 60 compounds were found to interact with the amino acid residues (GLU166, GLY143, ASP187, CYS145, GLN189, LEU141, etc.) which were critically involved in the 6LU7 domain mainly by hydrogen-bonded interaction. The network exploring results revealed that these potential compounds were mainly attributed to Glycyrrhizae Radix et Rhizoma, Mori Cortex, Rhododendron dauricum, Polygoni Cuspidati Rhizoma et Radix, and Plantaginis Herba, etc., which associates with acute lung syndromes induced by SARS-CoV-2, with the effect of clearing heat and removing toxin, relieving cough and dispelling phlegm and lung-draining and relieving asthma. Conclusion: Molecular docking method provides a useful tool for the screening of SARS-CoV-2 3CL protease inhibitors from TCMSP platform.

Exploring active compounds of Jinhua Qinggan Granules for prevention of COVID-19 based on network pharmacology and molecular docking / 中草药

Objective: To explore the effective chemical constituents of Jinhua Qinggan Granules for treatment of coronavirus disease 2019 (COVID-19). Methods: The compounds and action targets of eleven herbal medicines in Jinhua Qinggan Granules were collected via TCMSP. The genes corresponding to the targets were queried by the UniProt database, then the “herbal medicine-compound-target” network was established by Cytoscape software. The gene ontology (GO) function enrichment analysis and KEGG pathway enrichment analysis were performed by DAVID to predict their mechanism. Molecular docking was used to analyze the binding force of the core effective compounds in the “herbal medicine-compound-target” network with SARS-CoV-2 3CL hydrolase and angiotensin converting enzyme II (ACE2). Results: The “herbal medicine-compound-target” network contained 154 compounds and 276 targets, and the key targets involved PTGS2, HSP90AB1, HSP90AA1, PTGS1, NCOA2, etc. GO function enrichment analysis revealed 278 items, including ATP binding, transcription factor activation and regulation of apoptosis process, etc. KEGG pathway enrichment screened 127 signaling pathways, including TNF, PI3K/Akt and HIF-1 signaling pathways related to lung injury protection. The results of molecular docking showed that formononetin, stigmasterol, beta-sitosterol, anhydroicaritin and other key compounds have a certain degree of affinity with SARS-CoV-2 3CL hydrolase and ACE2. Conclusion: The effective compounds in Jinhua Qinggan Granules regulate multiple signaling pathways via binding ACE2 and acting on targets such as PTGS2, HSP90AB1, HSP90AA1, PTGS1, NCOA2 for the prevention of COVID-19.