RESUMEN
In the pursuit of novel antiretroviral therapies for human immunodeficiency virus type-1 (HIV-1) proteases (PRs), recent improvements in drug discovery have embraced machine learning (ML) techniques to guide the design process. This study employs ensemble learning models to identify crucial substructures as significant features for drug development. Using molecular docking techniques, a collection of 160 darunavir (DRV) analogs was designed based on these key substructures and subsequently screened using molecular docking techniques. Chemical structures with high fitness scores were selected, combined, and one-dimensional (1D) screening based on beyond Lipinski's rule of five (bRo5) and ADME (absorption, distribution, metabolism, and excretion) prediction implemented in the Combined Analog generator Tool (CAT) program. A total of 473 screened analogs were subjected to docking analysis through convolutional neural networks scoring function against both the wild-type (WT) and 12 major mutated PRs. DRV analogs with negative changes in binding free energy ( ΔΔ G bind ) compared to DRV could be categorized into four attractive groups based on their interactions with the majority of vital PRs. The analysis of interaction profiles revealed that potent designed analogs, targeting both WT and mutant PRs, exhibited interactions with common key amino acid residues. This observation further confirms that the ML model-guided approach effectively identified the substructures that play a crucial role in potent analogs. It is expected to function as a powerful computational tool, offering valuable guidance in the identification of chemical substructures for synthesis and subsequent experimental testing.
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Infecciones por VIH , Inhibidores de la Proteasa del VIH , VIH-1 , Humanos , Darunavir/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Inhibidores de la Proteasa del VIH/química , Péptido Hidrolasas/farmacología , Simulación del Acoplamiento Molecular , Proteasa del VIH/química , Descubrimiento de DrogasRESUMEN
A novel method for synthesizing cationic styryl dyes through a nucleic acid-templated reaction has been developed. This approach overcomes issues associated with traditional synthesis methods, such as harsh conditions, low throughput, and wasteful chemicals. The presence of a nucleic acid template accelerated the styryl dye formation from quaternized heteroaromatic and cationic aldehyde substrates. These styryl dyes show remarkable optical properties change when bound to nucleic acids, hence the success of the synthesis could be readily monitored inâ situ by UV-Vis and fluorescence spectroscopy and the optical properties data were also observable at the same time. This method provides the desired products from a broad range of coupling partners. By employing different substrates and templates, it is possible to identify new dyes that can bind to a specific type of nucleic acid such as a G-quadruplex. The templated dye synthesis is also successfully demonstrated in live HeLa cells. This approach is a powerful tool for the rapid synthesis and screening of dyes specific for diverse types of nucleic acids or cellular organelles, facilitating new biological discoveries.
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Cationes , Colorantes Fluorescentes , Ácidos Nucleicos , Humanos , Células HeLa , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Ácidos Nucleicos/química , Ácidos Nucleicos/síntesis química , Cationes/química , Espectrometría de Fluorescencia , G-Cuádruplex , ADN/química , Estirenos/química , Estirenos/síntesis química , Colorantes/química , Colorantes/síntesis químicaRESUMEN
The Asp-tRNAAsn/Glu-tRNAGln amidotransferase (GatCAB) has been proposed as a novel antibacterial drug target due to its indispensability in prominent human pathogens. While several inhibitors with in vitro activity have been identified, none have been demonstrated to have potent activity against live bacteria. In this work, seven non-hydrolyzable transition state mimics of GatCAB were synthesized and tested as the transamidase inhibitors against GatCAB from the human pathogen Helicobacter pylori. Notably, the methyl sulfone analog of glutamyl-adenosine significantly reduced GatCAB's transamination rate. Additionally, four lipid-conjugates of these mimics displayed antibacterial activity against Bacillus subtilis, likely due to enhanced cell permeability. Inhibitory activity against GatCAB in live bacteria was confirmed using a sensitive gain-of-function dual luciferase reporter in Mycobacterium bovis-BCG. Only the lipid-conjugated methyl sulfone analog exhibited a significant increase in mistranslation rate, highlighting its cell permeability and inhibitory potential. This study provides insights for developing urgently needed novel antibacterial agents amidst emerging antimicrobial drug resistance.
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Antibacterianos , Inhibidores Enzimáticos , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Relación Estructura-Actividad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Bacillus subtilis/efectos de los fármacos , Estructura Molecular , Relación Dosis-Respuesta a Droga , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/química , Adenosina/síntesis química , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/enzimología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Transferasas de Grupos Nitrogenados/metabolismo , HumanosRESUMEN
BACKGROUND: To investigate the effect of a 50% ascorbic acid with 50% citric acid solution on the immediate shear bond strength (SBS) of metallic brackets after tooth bleaching. The enamel etching pattern and the required quantity of these combined acids as antioxidants following 35% hydrogen peroxide (HP) bleaching were also determined. METHODS: The stability of the solution at room temperature was assessed at various time intervals. Fifty teeth were randomly divided into five groups: non-bleached (G1), bleached then acid etched (G2), bleached followed by a 10-minute treatment with 10% sodium ascorbate and acid etched (G3), 5-minute treatment with 50% ascorbic acid (G4), and 5-minute treatment with a combination of 50% ascorbic acid and 50% citric acid (G5). Groups G2, G3, G4 and G5 were bleached by 35% HP gel for a total of 32 min. Acid etching in groups G1, G2, and G3 was performed using 37% phosphoric acid (Ormco®, Orange, CA, USA) for 15 s. In all groups, metal brackets were immediately bonded using Transbond™ XT primer and Transbond™ PLUS adhesive, with light curing for 40 s. The SBS was tested with a universal testing machine, and statistical analysis was conducted using one-way ANOVA followed by Tukey's HSD test. The level of significance was set at p < 0.05 for all statistical tests. RESULTS: Stability tests demonstrated that the combined acids remained effective for up to 21 days. Group G5 significantly increased the SBS of bleached teeth to the level of G1 (p < 0.05), while G3 did not achieve the same increase in SBS (p > 0.05). SEM analysis revealed enamel etching patterns similar to those of both control groups (G1 and G2). Kinetic studies at 6 min indicated that the antioxidation in G5 reacted 0.2 mmole lower than in G3 and G4. CONCLUSION: 5-minute application of the combined acids enhanced the SBS of bleached teeth comparable to unbleached teeth. The combined acids remain stable over two weeks, presenting a time-efficient, single-step solution for antioxidant application and enamel etching in orthodontic bracket bonding.
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Ácido Ascórbico , Ácido Cítrico , Recubrimiento Dental Adhesivo , Esmalte Dental , Soportes Ortodóncicos , Resistencia al Corte , Blanqueamiento de Dientes , Ácido Ascórbico/farmacología , Ácido Cítrico/farmacología , Ácido Cítrico/química , Blanqueamiento de Dientes/métodos , Humanos , Proyectos Piloto , Esmalte Dental/efectos de los fármacos , Recubrimiento Dental Adhesivo/métodos , Grabado Ácido Dental , Antioxidantes/farmacología , Propiedades de Superficie , Factores de Tiempo , Peróxido de Hidrógeno/química , Blanqueadores Dentales/química , Ácidos Fosfóricos , Análisis del Estrés DentalRESUMEN
O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.
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Antígenos de Neoplasias/metabolismo , Histona Acetiltransferasas/metabolismo , Hialuronoglucosaminidasa/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Dominio Único/química , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Bioensayo , Dominio Catalítico , Sistemas de Liberación de Medicamentos/métodos , Expresión Génica , Glicosilación , Células HEK293 , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Humanos , Hialuronoglucosaminidasa/química , Hialuronoglucosaminidasa/genética , Hidrólisis , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Glicoproteínas de Membrana/genética , Proteínas de Complejo Poro Nuclear/genética , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Dominio Único/metabolismo , Factor de Transcripción Sp1/genética , Factores de Transcripción/genética , Transfección/métodosRESUMEN
Light-emitting plants (LEPs) provides light in areas without electricity. The phosphorescent compound was used as a lighting material for LEP development. However, using the phosphorescent compound for LEPs development required optimization and phytotoxicity evaluation. Strontium aluminate (SrAl2 O4 ) is a phosphorescent compound that can glow for a long time and is easily recharged by visible light. In this study, using SrAl2 O4 to develop LEPs was evaluated. Additionally, plant stress under SrAl2 O4 was investigated. Metabolomic analysis can explain the possible mechanism of plants' stress under SrAl2 O4 . After, injecting 3â mL of 5 % (w/v) SrAl2 O4 products 1, 2, and 3 into the stem of Ipomoea aquatica, the result showed that SrAl2 O4 products 2 and 3 caused oxidative stress. The metabolomic analysis also indicated that I.â aquatica responded to SrAl2 O4 product 1 by increasing pipecolic acid and salicylic acid, while I.â aquatica injected with SrAl2 O4 products 2 and 3 showed a decrease in salicylic acid around 0.005 and 0.061-fold, respectively, compared to control plants. and an excess accumulation of MDA around 10.00-12.00â µmol g-1 FW. A 15 % concentration of SrAl2 O4 can be used for LEPs development, enabling photoemission 18-fold for 50â min. SrAl2 O4 product 1 has the potential to be a material for LEPs.
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Luz , Estroncio , Desarrollo de la PlantaRESUMEN
Background and Objectives: Natural products have proven to be a valuable source for the discovery of new candidate drugs for cancer treatment. This study aims to investigate the potential therapeutic effects of "Kerra™", a natural extract derived from a mixture of nine medicinal plants mentioned in the ancient Thai scripture named the Takxila Scripture, on HCT116 cells. Materials and Methods: In this study, the effect of the Kerra™ extract on cancer cells was assessed through cell viability assays. Apoptotic activity was evaluated by examining the apoptosis characteristic features. A proteomics analysis was conducted to identify proteins and pathways associated with the extract's mechanism of action. The expression levels of apoptotic protein markers were measured to validate the extract's efficacy. Results: The Kerra™ extract demonstrated a dose-dependent inhibitory effect on the cells, with higher concentrations leading to decreased cell viability. Treatment with the extract for 72 h induced characteristic features of early and late apoptosis, as well as cell death. An LC-MS/MS analysis identified a total of 3406 proteins. The pathway analysis revealed that the Kerra™ extract stimulated apoptosis and cell death in colorectal cancer cell lines and suppressed cell proliferation in adenocarcinoma cell lines through the EIF2 signaling pathway. Upstream regulatory proteins, including cyclin-dependent kinase inhibitor 1A (CDKN1A) and MYC proto-oncogene, bHLH transcription factor (MYC), were identified. The expressions of caspase-8 and caspase-9 were significantly elevated by the Kerra™ extract compared to the chemotherapy drug Doxorubicin (Dox). Conclusions: These findings provide strong evidence for the ability of the Kerra™ extract to induce apoptosis in HCT116 colon cancer cells. The extract's efficacy was demonstrated by its dose-dependent inhibitory effect, induction of apoptotic activity, and modulation of key proteins involved in cell death and proliferation pathways. This study highlights the potential of Kerra™ as a promising therapeutic agent in cancer treatment.
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Antineoplásicos , Células HCT116 , Extractos Vegetales , Proteómica , Cromatografía Liquida , Células HCT116/efectos de los fármacos , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Espectrometría de Masas en Tándem , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Tailandia , Medicina TradicionalRESUMEN
O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.
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N-Acetilglucosaminiltransferasas/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico/análisis , Ácido Aspártico/metabolismo , Dominio Catalítico , Glicosilación , Humanos , Modelos Moleculares , N-Acetilglucosaminiltransferasas/química , Especificidad por Sustrato , Repeticiones de TetratricopéptidosRESUMEN
Kinetochores act as hubs for multiple activities during cell division, including microtubule interactions and spindle checkpoint signaling. Each kinetochore can act autonomously, and activities change rapidly as proteins are recruited to, or removed from, kinetochores. Understanding this dynamic system requires tools that can manipulate kinetochores on biologically relevant temporal and spatial scales. Optogenetic approaches have the potential to provide temporal and spatial control with molecular specificity. Here we report new chemical inducers of protein dimerization that allow us to both recruit proteins to and release them from kinetochores using light. We use these dimerizers to manipulate checkpoint signaling and molecular motor activity. Our findings demonstrate specialized properties of the CENP-E (kinesin-7) motor for directional chromosome transport to the spindle equator and for maintenance of metaphase alignment. This work establishes a foundation for optogenetic control of kinetochore function, which is broadly applicable to experimental probing of other dynamic cellular processes.
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Cinetocoros/metabolismo , Optogenética/métodos , Supervivencia Celular , Células HeLa , Humanos , Cinetocoros/química , Células Tumorales CultivadasRESUMEN
We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.
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Dimerización , Endopeptidasas/química , Tetrahidrofolato Deshidrogenasa/química , Compartimento Celular/efectos de los fármacos , Compartimento Celular/genética , Compartimento Celular/efectos de la radiación , Luz , Orgánulos/química , Orgánulos/efectos de la radiación , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Transporte de Proteínas/efectos de la radiación , Sirolimus/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tetrahidrofolato Deshidrogenasa/efectos de la radiaciónRESUMEN
Many dynamic biological processes are regulated by protein-protein interactions and protein localization. Experimental techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chemically induced dimerization (CID) of proteins. CID has been used to study several cellular events, especially cell signaling networks, which are often reversible. However, chemical dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resolution are currently limited. Herein, we present a novel chemical inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this molecule by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biological questions on subcellular levels by controlling protein-protein interactions.
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Proteínas Bacterianas/metabolismo , Cumarinas/química , Indicadores y Reactivos/química , Trimetoprim/química , Proteínas Bacterianas/química , Cumarinas/toxicidad , Diseño de Fármacos , Escherichia coli/enzimología , Células HeLa , Humanos , Indicadores y Reactivos/toxicidad , Cinetocoros/metabolismo , Listeria monocytogenes/química , Mitocondrias/metabolismo , Peroxisomas/metabolismo , Multimerización de Proteína , Rhodococcus/enzimología , Trimetoprim/toxicidad , Rayos UltravioletaRESUMEN
BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe form of lung injury characterized by damage to the epithelial barrier with subsequent pulmonary edema and hypoxic respiratory failure. ARDS is a significant medical problem in intensive care units with associated high care costs. There are many potential causes of ARDS; however, alveolar injury associated with mechanical ventilation, termed ventilator-induced lung injury (VILI), remains a well-recognized contributor. It is thus critical to understand the mechanism of VILI. Based on our published preliminary data, we hypothesized that the endoplasmic reticulum (ER) stress response molecule Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) plays a role in transmitting mechanosensory signals the alveolar epithelium. METHODS: ER stress signal responses to mechanical stretch were studied in ex-vivo ventilated pig lungs. To explore the effect of PERK inhibition on VILI, we ventilated live rats and compared lung injury parameters to non-ventilated controls. The effect of stretch-induced epithelial ER Ca2+ signaling on PERK was studied in stretched alveolar epithelial monolayers. To confirm the activation of PERK in human disease, ER stress signaling was compared between ARDS and non-ARDS lungs. RESULTS: Our studies revealed increased PERK-specific ER stress signaling in response to overstretch. PERK inhibition resulted in dose-dependent improvement of alveolar inflammation and permeability. Our data indicate that stretch-induced epithelial ER Ca2+ release is an activator of PERK. Experiments with human lung tissue confirmed PERK activation by ARDS. CONCLUSION: Our study provides evidences that PERK is a mediator stretch signals in the alveolar epithelium.
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Estrés del Retículo Endoplásmico/fisiología , Pulmón/metabolismo , Receptores de Estiramiento Pulmonares/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , eIF-2 Quinasa/fisiología , Adulto , Anciano , Animales , Femenino , Humanos , Pulmón/patología , Masculino , Persona de Mediana Edad , Receptores de Estiramiento Pulmonares/patología , Ratas , Ratas Sprague-Dawley , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Porcinos , Lesión Pulmonar Inducida por Ventilación Mecánica/patologíaRESUMEN
The prevalence of HIV-1 infection continues to pose a significant global public health issue, highlighting the need for antiretroviral drugs that target viral proteins to reduce viral replication. One such target is HIV-1 protease (PR), responsible for cleaving viral polyproteins, leading to the maturation of viral proteins. While darunavir (DRV) is a potent HIV-1 PR inhibitor, drug resistance can arise due to mutations in HIV-1 PR. To address this issue, we developed a novel approach using the fragment molecular orbital (FMO) method and structure-based drug design to create DRV analogs. Using combinatorial programming, we generated novel analogs freely accessible via an on-the-cloud mode implemented in Google Colab, Combined Analog generator Tool (CAT). The designed analogs underwent cascade screening through molecular docking with HIV-1 PR wild-type and major mutations at the active site. Molecular dynamics (MD) simulations confirmed the assess ligand binding and susceptibility of screened designed analogs. Our findings indicate that the three designed analogs guided by FMO, 19-0-14-3, 19-8-10-0, and 19-8-14-3, are superior to DRV and have the potential to serve as efficient PR inhibitors. These findings demonstrate the effectiveness of our approach and its potential to be used in further studies for developing new antiretroviral drugs.
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Infecciones por VIH , Inhibidores de la Proteasa del VIH , VIH-1 , Humanos , Darunavir/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Inhibidores de la Proteasa del VIH/química , VIH-1/genética , Simulación del Acoplamiento Molecular , Sulfonamidas/farmacología , Proteínas Virales/genética , Proteasa del VIH/metabolismo , Mutación , Farmacorresistencia Viral/genéticaRESUMEN
Reactive oxygen species (ROS) play a critical role in oxidative stress and cellular damage, underscoring the importance of identifying potent antioxidants. This research focuses on the antioxidant capabilities of Riceberry™-derived peptides and their protective effects against oxidative and endoplasmic reticulum (ER) stress in L929 cells. By simulating human digestion, Riceberry™ protein hydrolysate was generated, from which antioxidant peptides were isolated using OFFGEL electrophoresis and LC-MS/MS. Notably, an octapeptide (VPAGVAHW) from the hydrolysate demonstrated significant antioxidant activity, particularly against oxidative stress induced by iodoacetic acid (IAA) or hydrogen peroxide (H2O2) and ER stress caused by tunicamycin (TM) in L929 cells. This peptide's effectiveness was evident in its dose-dependent ability to enhance cell viability and mitigate stress effects, although its efficiency varied with the stress inducer. Our study suggests that Riceberry™-derived peptides could serve as a promising natural antioxidant with potential benefits for health promotion and applications in the food industry, offering an environmentally friendly alternative to synthetic antioxidants.
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One new alkyl benzoquinone, paphionone (1), one new trans-stilbenoid, (E)-6,5'-dihydroxy-2,3'-dimethoxystilbene (2), and eight known stilbenoids and flavonoids (3-10) were isolated from the leaves and roots of Paphiopedilum exul (Orchidaceae). Their chemical structures were determined based on IR, ECD, MS and NMR analyses. Cytotoxicity of all isolated compounds towards human hepatocellular carcinoma (HepG2) cell line was examined in vitro by MTT assay. The para-hydroxybenzyl substituted stilbene 10 was potently cytotoxic to the cancer cells, with an IC50 value of 4.80 ± 1.10 µM (selectivity index = 20.83). All compounds were non-toxic to normal human embryo fibroblast (OUMS-36) cell line.
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Tight junction disruption can lead to pathogenesis of various diseases without therapeutic strategy to recover intestinal barrier integrity. The main objective of this study is to demonstrate the effect of Solanum melongena L. extract (SMLE) on intestinal tight junction recovery and its underlying mechanism. Intestinal barrier function is attenuated by Ca2+ depletion. SMLE treatment increased TER value across T84 cell monolayers. Permeability assay reveals that Ca2+ depletion promotes 4-kDa FITC-dextran permeability, but not 70-kDa FITC-dextran. SMLE suppresses the rate of 4-kDa FITC-dextran permeability, indicating that SMLE inhibits paracellular leak pathway permeability. SMLE-mediated TER increase and leak pathway suppression are abolished by neither calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor nor AMP-activated protein kinase (AMPK) inhibitor. Furthermore, mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) inhibitors have no effects on SMLE-mediated TER increase and leak pathway suppression. Interestingly, SMLE is unable to enhance TER value and diminish leak pathway permeability in T84 cell monolayers pre-treated with sirtuin-1 (SIRT-1) inhibitor. Immunofluorescence staining reveals that SMLE enhances re-assembly of tight junction proteins, including occludin and ZO-1 to intercellular space but this effect is abolished by SIRT-1 inhibitor. These data suggest that SMLE promotes intestinal tight junction re-assembly via SIRT-1-dependent manner.
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Purpose: Cultured lichen mycobionts are valuable sources of new natural compounds. Mycobiont of Graphis handelii growing in Vietnam was isolated, cultivated and chemically investigated. The crude extract of this cultured mycobiont showed potent alpha-glucosidase inhibition with an IC50 value of 50 µg/mL. Methods: Multiple chromatographic methods were applied to the extract to isolate compounds. The combination of Nuclear Magnetic Resonance analysis and high-resolution mass spectroscopy determined their chemical structures. Electrophilic bromination/chlorination was applied to obtain new derivatives using NaBr/H2O2 and NaCl/H2O2 reagents. Compounds were evaluated for enzyme inhibitory activities, including alpha-glucosidase inhibition, HIV-1 reverse transcriptase inhibition, SARS-CoV-2 main protease (Mpro) inhibition, anti-inflammatory activity, and cytotoxicity against several cancer cell lines. A molecular docking study for anti-SARS-CoV-2 was conducted to understand the inhibitory mechanism. Results: A new diphenyl ether, handelone (1) and a known compound xylarinic acid A (2) were isolated and elucidated. Four synthetic products 6'-bromohandelone (1a), 2'-bromohandelone (1b), 2',6'-dibromohandelone (1c), and 2',6'-dichlorohandelone (1d) were prepared. Compound 1 showed good activity against Mpro with an IC50 value of 5.2 µM but it showed weak or inactive activity in other tests. Other compounds were inactive in all assays. Conclusion: A new compound, handelone (1) was isolated from the cultured mycobiont of Graphis handelii. From these compounds, four new derivatives were prepared. Compound 1 showed good activity against Mpro with an IC50 value of 5.2 µM but it showed weak or inactive activity in other tests. Other compounds were inactive in all assays.
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Antibacterial proteins inhibiting Pseudomonas aeruginosa have been identified in various phages and explored as antibiotic alternatives. Here, we isolated a phiKZ-like phage, Churi, which encodes 364 open reading frames. We examined 15 early-expressed phage proteins for their ability to inhibit bacterial growth, and found that gp335, closely related to phiKZ-gp14, exhibits antibacterial activity. Similar to phiKZ-gp14, recently shown to form a complex with the P. aeruginosa ribosome, we predict experimentally that gp335 interacts with ribosomal proteins, suggesting its involvement in protein translation. GFP-tagged gp335 clusters around the phage nucleus as early as 15 minutes post-infection and remains associated with it throughout the infection, suggesting its role in protein expression in the cell cytoplasm. CRISPR-Cas13-mediated deletion of gp355 reveals that the mutant phage has a prolonged latent period. Altogether, we demonstrate that gp335 is an antibacterial protein of nucleus-forming phages that associates with the ribosomes at the phage nucleus.
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A series of novel styryl dye derivatives incorporating indolium and quinolinium core structures were successfully synthesized to explore their interacting and binding capabilities with tau aggregates inâ vitro and in cells. The synthesized dyes exhibited enhanced fluorescence emission in viscous environments due to the rotatable bond confinement in the core structure. Dye 4, containing a quinolinium moeity and featuring two cationic sites, demonstrated a 28-fold increase in fluorescence emission upon binding to tau aggregates. This dye could also stain tau aggregates in living cells, confirmed by cell imaging using confocal fluorescence microscopy. A molecular docking study was conducted to provide additional visualization and support for binding interactions. This work offers novel and non-cytotoxic fluorescent probes with desirable photophysical properties, which could potentially be used for studying tau aggregates in living cells, prompting further development of new fluorescent probes for early Alzheimer's disease detection.