RESUMEN
The microtubule-associated protein tau oligomerizes, but the actions of oligomeric tau (oTau) are unknown. We have used Cry2-based optogenetics to induce tau oligomers (oTau-c). Optical induction of oTau-c elicits tau phosphorylation, aggregation, and a translational stress response that includes stress granules and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau-c. The association of HNRNPA2B1 with endogenous oTau was verified in neurons, animal models, and human Alzheimer brain tissues. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine (m6A) modified RNA transcripts. Knockdown of HNRNPA2B1 prevents oTau or oTau-c from associating with m6A or from reducing protein synthesis and reduces oTau-induced neurodegeneration. Levels of m6A and the m6A-oTau-HNRNPA2B1 complex are increased up to 5-fold in the brains of Alzheimer subjects and P301S tau mice. These results reveal a complex containing oTau, HNRNPA2B1, and m6A that contributes to the integrated stress response of oTau.
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Adenosina/análogos & derivados , Enfermedad de Alzheimer/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Procesamiento Postranscripcional del ARN , ARN/metabolismo , Proteínas tau/metabolismo , Adenosina/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Masculino , Metilación , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Agregado de Proteínas , Agregación Patológica de Proteínas , ARN/genética , Índice de Severidad de la Enfermedad , Proteínas tau/genéticaRESUMEN
With the advent of mitochondrial targeting moiety such as triphenlyphosphonium cation (TPP+), targeting mitochondria in cancer cells has become a promising strategy for combating tumors. Herein, a series of novel 4-aryl-1,3-thiazole derivatives linked to TPP+ moiety were designed and synthesized. The cytotoxicity against a panel of four cancer cell lines was evaluated by CCK-8 assay. Most of these compounds exhibited moderate to good inhibitory activity over HeLa, PC-3 and HCT-15 cells while MCF-7 cells were less sensitive to most compounds. Among them, compound 12a exhibited a significant anti-proliferative activity against HeLa cells, and prompted for further investigation. Specifically, 12a decreased mitochondrial membrane potential and enhanced levels of reactive oxygen species (ROS). The flow cytometry analysis revealed that compound 12a could induce apoptosis and cell cycle arrest at G0/G1 phase in HeLa cells. In addition, mitochondrial bioenergetics assay revealed that 12a displayed mild mitochondrial uncoupling effect. Taken together, these findings suggest the therapeutic potential of compound 12a as an antitumor agent targeting mitochondria.
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Antineoplásicos , Apoptosis , Proliferación Celular , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Potencial de la Membrana Mitocondrial , Mitocondrias , Especies Reactivas de Oxígeno , Tiazoles , Humanos , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Relación Estructura-Actividad , Tiazoles/farmacología , Tiazoles/química , Tiazoles/síntesis química , Estructura Molecular , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Apoptosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , Compuestos Organofosforados/farmacología , Compuestos Organofosforados/química , Compuestos Organofosforados/síntesis químicaRESUMEN
Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.
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Agregado de Proteínas , Agregación Patológica de Proteínas , Multimerización de Proteína , Antígeno Intracelular 1 de las Células T/metabolismo , Proteínas tau/metabolismo , Amiloide/química , Amiloide/metabolismo , Humanos , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas con Motivos de Reconocimiento de ARN/química , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas Recombinantes , Proteínas tau/químicaRESUMEN
Mitochondria are pivotal energy production sources for cells to maintain necessary metabolism activities. Targeting dysfunctional mitochondrial features has been a hotspot for mitochondrial-related disease researches. Investigation with cancerous mitochondrial metabolism is a continuing concern within tumor therapy. Herein, we set out to assess the anti-cancer activities of a novel family of TPP-thiazole derivatives based on our earlier research on mitochondrial targeting agents. Specifically, we designed and synthesized a series of TPP-thiazole derivatives and revealed by the MTT assay that most synthesized compounds effectively inhibited three cancer cell lines (HeLa, PC3 and MCF-7). After structure modifications, we explored the SAR relationships and identified the most promising compound R13 (IC50 of 5.52 µM) for further investigation. In the meantime, we performed ATP production assay to assess the selected compounds inhibitory effect on HeLa cells energy production. The results displayed the test compounds significantly restrained ATP production of cancer cells. Overall, we have designed and synthesized a series of compounds which exhibited significant cytotoxicity against cancer cells and effectively inhibited mitochondrial energy production.
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Antineoplásicos/farmacología , Mitocondrias/efectos de los fármacos , Compuestos Organofosforados/farmacología , Tiazoles/farmacología , Adenosina Trifosfato/metabolismo , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Mitocondrias/metabolismo , Estructura Molecular , Compuestos Organofosforados/síntesis química , Relación Estructura-Actividad , Tiazoles/síntesis químicaRESUMEN
Cancer therapy targets specific metabolic pathways or a single gene. This may result in low therapeutic effects due to drug selectivity and drug resistance. Recent studies revealed that the mitochondrial membrane potential and transmembrane permeability of cancerous mitochondria are differed from normal mitochondria. Thus, chemotherapy targeting cancerous mitochondria could be an innovative and competent strategy for cancer therapy. Previously, our work with a novel group of mitochondria targeting small molecules presented promising inhibitory capability toward various cancer cell lines and suppressed adenosine triphosphate (ATP) generation. Therefore, it is critical to understand the anticancer effect and targeting mechanism of these small molecules. This study investigated the inhibitory activity of mitochondria targeting small molecules with human cervical cancer cells - HeLa to further explore their therapeutic potential. HeLa cells were exposed to 10 µM of synthesized compounds and presented elevation in intracellular reactive oxygen species (ROS) level, impaired mitochondrial membrane potential and upregulation of apoptosis as well as necrosis. In vivo, HeLa cell tumor-bearing BALB/c nude mice were treated with mitochondria targeting small molecules for 12 days consecutively. Throughout this chemotherapy study, no deleterious side effects nor the appearance of toxicity was observed. Furthermore, mitochondria targeting small molecules treated groups exhibited significant down-regulation with both tumor volume and tumor weight compared to the Doxorubicin (DOX) treated group. Thus, inhibition of mitochondrial ATP synthesis, activation of intracellular ROS production, down-regulation of mitochondrial membrane potential and upregulation of apoptosis and necrosis rates are the indications of cancer therapy. In this work, we examined the anticancer capability of four mitochondria targeting small molecules in vitro and in vivo, and demonstrated a novel therapeutic approach in cancer therapy with tremendous potential.
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Antineoplásicos/farmacología , Mitocondrias/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Tiazoles/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mitocondrias/metabolismo , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/químicaRESUMEN
Incretin pathway plays an important role in the development of diabetes medications. Interventions in DPP-4 and GLP-1 receptor have shown remarkable efficacy in experimental and clinical studies and imperatively become one of the most promising therapeutic approaches in the T2DM drug discovery pipeline. Herein, we analyzed the actionmechanismsof DPP-4 and GLP-1 receptor targeting the incretin pathway in T2DM treatment. We gave an insight into the structural requirements for the potent DPP-4 inhibitors and revealed a classification of DPP-4 inhibitors by stressing on the binding modes of these ligands to the enzyme. We then reviewed the drug discovery strategies for the development of peptide and non-peptide GLP-1 receptor agonists (GLP-1 RAs). Furthermore, the drug design strategies for DPP-4 inhibitors and GLP-1R agonists were detailed accurately. This review might provide an efficient evidence for the highly potent and selective DPP-4 inhibitors and the GLP-1 RAs, as novel medicines for patients suffering from T2DM.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Descubrimiento de Drogas , Hipoglucemiantes/farmacología , Incretinas/antagonistas & inhibidores , Péptidos/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/química , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Hipoglucemiantes/química , Incretinas/metabolismo , Modelos Moleculares , Péptidos/químicaRESUMEN
BACKGROUND: Theabrownin (TB) is a dark brown pigment from Pu-erh tea or other dark teas. It is formed by further oxidization of theaflavins and thearubigins, in combination with proteins, polysaccharides, and caffeine etc. TB is a characteristic ingredient and bioactive substance of Pu-erh tea. However, the effects of TB on ulcerative colitis (UC) remains unclear. PURPOSE: This study aims to elucidate the mechanism of TB on UC in terms of recovery of intestinal homeostasis and regulation of toll-like receptor (TLR) 2&4 signaling pathway. METHODS: The colitis models were established by administering 5% dextran sulfate sodium (DSS) to C57BL/6 mice for 5 days to evaluate the therapeutic and preventive effects of TB on UC. Mesalazine was used as a positive control. H&E staining, complete blood count, enzyme-linked immunosorbent assay, immunohistochemistry, flow cytometry, and 16S rRNA sequencing were employed to assess histological changes, blood cells analysis, content of cytokines, expression and distribution of mucin (MUC)2 and TLR2&4, differentiation of CD4+T cells in lamina propria, and changes in intestinal microbiota, respectively. Western blot was utilized to study the relative expression of tight junction proteins and the key proteins in TLR2&4-mediated MyD88-dependent MAPK, NF-κB, and AKT signaling pathways. RESULTS: TB outstanding alleviated colitis, inhibited the release of pro-inflammatory cytokines, reduced white blood cells while increasing red blood cells, hemoglobin, and platelets. TB increased the expression of occludin, claudin-1 and MUC2, effectively restored intestinal barrier function. TB also suppressed differentiation of Th1 and Th17 cells in the colon's lamina propria, increased the fraction of Treg cells, and promoted the balance of Treg/Th17 to tilt towards Tregs. Moreover, TB increased the Firmicutes to Bacteroides (F/B) ratio, as well as the abundance of Akkermansia, Muribaculaceae, and Eubacterium_coprostanoligenes_group at the genus level. In addition, TB inhibited the activation of TLR2&4-mediated MAPK, NF-κB, and AKT signaling pathways in intestinal epithelial cells of DSS-induced mice. CONCLUSION: TB acts in restoring intestinal homeostasis and anti-inflammatory in DSS-induced UC, and exhibiting a preventive effect after long-term use. In a word, TB is a promising beverage, health product and food additive for UC.
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Sulfato de Dextran , Homeostasis , Ratones Endogámicos C57BL , Transducción de Señal , Té , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Animales , Receptor Toll-Like 2/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Té/química , Homeostasis/efectos de los fármacos , Ratones , Masculino , Catequina/farmacología , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/efectos de los fármacos , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/inducido químicamente , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Citocinas/metabolismo , FN-kappa B/metabolismoRESUMEN
Background: Although caffeine generally offers benefits to human health, its impact on bone metabolism remains unclear. Aim and Methods: This study aimed to systematically evaluate the long-term effects of caffeine administration on osteoclasts, osteoblasts, and ovariectomy-induced postmenopausal osteoporosis (OP). Results: Our in vitro findings revealed that 3.125 and 12.5 µg/mL caffeine inhibited RANKL-mediated osteoclastogenesis in RAW 264.7 cells through the MAPK and NF-κB pathways, accompanied by the inactivation of nuclear translocation of nuclear factor NFATc1. Similarly, 3.125 and 12.5 µg/mL of caffeine modulated MC3T3-E1 osteogenesis via the AKT, MAPK, and NF-κB pathways. However, 50 µg/mL of caffeine promoted the phosphorylation of IκBα, P65, JNK, P38, and AKT, followed by the activation of NFATc1 and the inactivation of Runx2 and Osterix, ultimately disrupting the balance between osteoblastogenesis and osteoclastogenesis. In vivo studies showed that gavage with 55.44 mg/kg caffeine inhibited osteoclastogenesis, promoted osteogenesis, and ameliorated bone loss in ovariectomized mice. Conclusion: Conversely, long-term intake of high-dose caffeine (110.88 mg/kg) disrupted osteogenesis activity and promoted osteoclastogenesis, thereby disturbing bone homeostasis. Collectively, these findings suggest that a moderate caffeine intake (approximately 400 mg in humans) can regulate bone homeostasis by influencing both osteoclasts and osteoblasts. However, long-term high-dose caffeine consumption (approximately 800 mg in humans) could have detrimental effects on the skeletal system.
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Background: Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, but little is known regarding PTC metabolic phenotypes and the effects of mitochondrial activity on PTC progression. The great potential of mitochondria-targeting therapy in cancer treatment promoted us to use tool compounds from a family of Mito-Fu derivatives to investigate how the regulation of mitochondrial respiration affected tumor progression characteristics and molecular changes in PTC. Methods: Mito-Fu L20, a representative of 12 synthetic derivatives, was chosen for mitochondrial inhibition experiments. Sample sections from PTC patients were collected and processed to explore potential molecular alterations in tumor lymph node metastasis (LNM). In vitro analyses were performed using human PTC cell lines (K1 and TPC-1), with the human normal thyroid follicular cell line (Nthy) as a control. K1 cells were injected into nude mice to generate an animal model. The mice were injected with normal saline or Mito-Fu L20 at 20 or 50 mg/kg every other day; their body weights and tumor volumes were also measured over time. To elucidate the resulting metabolic phenotype, we measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), cellular adenosine triphosphate (ATP) levels and reactive oxygen species (ROS) production, and mitochondrial membrane potential. Wound healing and Transwell assays, cell cycle assays, real-time fluorescence quantitative PCR, Western blotting, and immunohistochemical staining were performed to explore glycolysis-dominant metabolism in PTC. Results: Cyclin D1 and mitochondrial complex IV were detected in tumor samples from PTC patients with LNM. Mito-Fu L20 showed dose-independent and reversible modulation of mitochondrial respiration in PTC. In addition to mitochondrial dysfunction and early apoptosis, G1/S phase arrest. Notably, reversible mitochondrial inhibition yielded durable suppression of tumor proliferation, migration, and invasion via the PI3K/Akt/FoxO1/Cyclin D1 pathway. In vivo experiments demonstrated that Mito-Fu L20 has a good safety profile and specific restorative effect on mitochondrial activity in the liver. In addition, Mito-Fu L20 showed antitumor effects, alleviated tumor angiogenesis, and improved thyroid function. Conclusion: Reversible inhibition of ATP production and durable suppression of PTC growth indicates that the downregulation of mitochondrial function has a negative impact on tumor progression and LNM via the PI3K/Akt/FoxO1/Cyclin D1 pathway. The results provide new insights into the antitumor potential and clinical translation of mitochondrial inhibitors.
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Sortase A (SrtA) is a cysteine transpeptidase of most gram-positive bacteria that is responsible for the anchoring of many surface protein virulence factors to the cell wall. SrtA ablation has demonstrated to alleviate the infection without affecting the viability of bacteria. Herein, a series of benzofuran cyanide derivatives were synthesized and evaluated. Several compounds exhibited excellent inhibitory activity against SrtA with IC50 values from 3.3 µM to 21.8 µM compared with the known SrtA inhibitor pHMB (IC50 = 130 µM). â ¢-1, â ¢-15, â ¢-34 and V-1 showed potent inhibitory effects on biofilm formation with IC50 values from 2.1 µM to 54.2 µM. Invasion assays showed the four compounds caused a decrease of 4%-24.0% in the uptake of the S. aureus strain by 293T cells. Further assay showed that compound â ¢-15 decreased the amount of cell wall-associated protein A by 26.5%. Structure-activity relationship and docking studies demonstrated that the acrylonitrile moiety of the compounds played an important role in enhancing the activity. When the double bond of acrylonitrile changed to single bond, the activity was decreased significantly. This indicates that acrylonitrile, which is a Michael receptor, can inhibit the activity of SrtA by covalent binding effectively to the thiol group of Cys184.
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Aminoaciltransferasas/antagonistas & inhibidores , Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Benzofuranos/química , Cianuros/química , Aminoaciltransferasas/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biopelículas/efectos de los fármacos , Cianuros/metabolismo , Cisteína Endopeptidasas/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Relación Estructura-ActividadRESUMEN
Cancer is a leading cause of death worldwide. Recent research studies have revealed that GM3 derivatives have considerable promise as potential therapeutic agents for cancer. To discover novel GM3 derivatives as potential antitumor agents, a one-pot enzymatic synthesis was established, yielding 14 GM3 derivatives in high total yields (22-41%). Subsequently, the inhibitory activities of GM3 derivatives were assessed by wound-healing assays and Transwell assays and tumor-bearing animal models. Among all the GM3 derivatives, N-12 showed excellent migration and invasion inhibitory effects in cells and marked antitumor activity in C57BL/6 mice. The subsequent analysis of cancer tissues and serum samples revealed that N-12 induces tumor inhibition, which was closely related to immune response. Taken together, N-12 can be further developed as an effective therapeutic for the treatment of cancer. An RNA-sequencing (RNA-seq) analysis was then performed and indicated that the antitumor mechanism of N-12 involved focal adhesion and ECM-receptor interaction signaling pathways.
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Antineoplásicos/uso terapéutico , Gangliósido G(M3)/análogos & derivados , Gangliósido G(M3)/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Gangliósido G(M3)/síntesis química , Gangliósido G(M3)/farmacocinética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunidad/efectos de los fármacos , Inmunoterapia , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Necrosis/inducido químicamente , Transducción de Señal/efectos de los fármacosRESUMEN
Neurodegeneration is a pathological condition in which nervous system or neuron losses its structure, function, or both leading to progressive neural degeneration. Growing evidence strongly suggests that reduction of plasmalogens (Pls), one of the key brain lipids, might be associated with multiple neurodegenerative diseases, including Alzheimer's disease (AD). Plasmalogens are abundant members of ether-phospholipids. Approximately 1 in 5 phospholipids are plasmalogens in human tissue where they are particularly enriched in brain, heart and immune cells. In this study, we employed a scheme of 2-months Pls intragastric administration to aged female C57BL/6J mice, starting at the age of 16 months old. Noticeably, the aged Pls-fed mice exhibited a better cognitive performance, thicker and glossier body hair in appearance than that of aged control mice. The transmission electron microscopic (TEM) data showed that 2-months Pls supplementations surprisingly alleviate age-associated hippocampal synaptic loss and also promote synaptogenesis and synaptic vesicles formation in aged murine brain. Further RNA-sequencing, immunoblotting and immunofluorescence analyses confirmed that plasmalogens remarkably enhanced both the synaptic plasticity and neurogenesis in aged murine hippocampus. In addition, we have demonstrated that Pls treatment inhibited the age-related microglia activation and attenuated the neuroinflammation in the murine brain. These findings suggest for the first time that Pls administration might be a potential intervention strategy for halting neurodegeneration and promoting neuroregeneration.
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Pathogens pose a severe threat to food safety and human health. The traditional methods for pathogen detection can't meet the growing diagnosis and control need. Digital PCR (dPCR) attracts a considerable attention for its ability to absolutely quantify pathogens with features of high selectivity, simplicity, accuracy and rapidity. The dPCR technique that achieves absolute quantification based on end-point measurement without standard curve offers a guideline for further genetic analysis and molecular diagnosis. It could contribute to the quantification of low level of nucleic acid, early detection and timely prevention of pathogenic diseases. In this review, 1442 publications about dPCR were selected and the detections of various pathogens by dPCR were reviewed comprehensively, including viruses, bacteria, parasites and fungi. A number of examples are cited to illustrate that dPCR is a new powerful tool with desired accuracy, sensitivity, and reproducibility for quantification of different types of pathogens. Moreover, the benefits, challenges and future prospects of the dPCR were also highlighted in this review.
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Infecciones/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Animales , Bacterias/aislamiento & purificación , Microbiología de Alimentos , Parasitología de Alimentos , Hongos/aislamiento & purificación , Humanos , Parásitos/aislamiento & purificación , Reproducibilidad de los Resultados , Virus/aislamiento & purificaciónRESUMEN
A series of benzofuran piperidine derivatives were designed, synthesized and evaluated as multifunctional Aß antiaggregant to treat Alzheimer's disease (AD). In vitro results revealed that all of them are very good Aß antiaggregants and some of the compounds are potent acetylcholinesterase (AChE) inhibitors with moderate antioxidant property. Selected compounds were also tested for neuroprotection activity, LDH release, ATP production and inhibitory activity to prevent Aß peptides binding to the cell membrane. The different modifications introduced in the structure of our lead compound 3 (hAChE IC50 = 61 µM and self induced Aß 25-35 aggregation 45.45%), to increase its activity toward AD related targets. The most interesting multifunctional Aß antiaggregants were compounds 3a, 3h and 3i, highlighting 3h as potent Aß antiaggregant and good antiacetylholinesterase inhibitor (self induced Aß 25-35 aggregation 57.71% and hAChE IC50 = 21 µM), with good neuroprotective and antioxidant activity. In addition, these three most promising compounds prevent intracellular reactive oxygen species (ROS) formation and cell apoptosis induced by Aß25-35 peptides in SH-SY5Y cells. Molecular docking studies were also accomplished to understand the binding interaction of these compounds on Aß monomer, Aß fibril and AChE. Based on all data, compounds 3a, 3h and 3i were concluded as potent multifunctional Aß antiaggregant, useful candidate for the treatment of AD.
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Péptidos beta-Amiloides/antagonistas & inhibidores , Benzofuranos/farmacología , Inhibidores de la Colinesterasa/farmacología , Fármacos Neuroprotectores/farmacología , Piperidinas/farmacología , Acetilcolinesterasa/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Benzofuranos/síntesis química , Benzofuranos/química , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Piperidinas/síntesis química , Piperidinas/química , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/tratamiento farmacológico , Agregación Patológica de Proteínas/metabolismo , Relación Estructura-ActividadRESUMEN
Liquid-liquid phase separation (LLPS) brings together functionally related proteins through the intrinsic biophysics of proteins in a process that is driven by reducing free energy and maximizing entropy. The process of LLPS allows proteins to form structures, termed membrane-less organelles. These diverse, dynamic organelles are active in a wide range of processes in the nucleus, cytoplasm, mitochondria and synapse, and ranging from bacteria to plants to eukaryotes. RNA and DNA present long chained charged polymers that promote LLPS. Consequently, many RNA binding proteins (RBPs) and DNA binding proteins form membrane-less organelles. However, the highly concentrated phase separated state creates conditions that also promote formation of irreversible protein aggregates. Mutations in RNA and DNA binding proteins that increase the stability of irreversible aggregates also increase the accumulation of irreversible aggregates directly and from membrane-less organelles. Many of the RBPs that exhibit disease-linked mutations carry out cytoplasmic actions through stress granules, which are a pleiotropic type of RNA granule that regulates the translational response to stress. Phosphorylation and oligomerization of tau facilitates its interactions with RBPs and ribosomal proteins, affecting RNA translation; we propose that this is a major reason that tau becomes phosphorylated with stress. Persistent stress leads to the accumulation of irreversible aggregates composed of RBPs or tau, which then cause toxicity and form many of the hallmark pathologies of major neurodegenerative diseases. This pathophysiology ultimately leads to multiple forms of neurodegenerative diseases, the specific type of which reflects the temporal and spatial accumulation of different aggregating proteins.
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Enfermedades Neurodegenerativas/fisiopatología , Agregado de Proteínas , Animales , Fenómenos Biofísicos , Gránulos Citoplasmáticos/metabolismo , Humanos , Mutación/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , ARN/metabolismoRESUMEN
Vibrio parahaemolyticus is a significant seafood-borne pathogen, leading to serious acute gastrointestinal diseases worldwide. In this study, a reliable 4-plex droplet digital PCR (ddPCR) was successfully established and evaluated for the simultaneous detection of V. parahaemolyticus based on tlh, tdh, ureR, and orf8 in food samples using single intact cells. The targets tlh and ureR were labeled with 6-Carboxyfluorescein (FAM), and the targets tdh and orf8 were labeled with 5'-Hexachlorofluorescein (HEX). Due to reasonable proration of primers and probes corresponding into the two fluorescence channels of the ddPCR detecting platforms, the clearly separated 16 (24) clusters based on fluorescence amplitude were obtained. For better results, the sample hot lysis time and the cycle number were optimized. The results showed that the minimum number of "rain" and maximum fluorescence amplification were presented for precise detection in the condition of 25 min of the sample hot lysis time and 55 cycles. The sensitivity of this 4-plex ddPCR assay was 39 CFU/mL, which was in accordance with that of the conventional plate counting and was 10-fold sensitive than that of qPCR. In conclusion, the 4-plex ddPCR assay presented in this paper was a rapid, specific, sensitive, and accurate tool for the detection of V. parahaemolyticus including pandemic group strains and could be applied in the differentiation of V. parahaemolyticus in a wide variety of samples.
RESUMEN
Our previous discovery of pyrazolo [1,5-a]pyrimidin-7(4H)-one scaffold-based DPP-4 inhibitors yielded two potent compounds b2 (IC50 = 79 nM) and d1 (IC50 = 49 nM) but characterized by cytotoxicity. Herein, with scaffold hopping and fragment-based drug design strategies, highly potent and selective pyrazolo [1,5-a]pyrimidine DPP-4 inhibitors were found featured by reduced or diminished cytotoxicity. Specifically, c24 (IC50 = 2 nM) exhibits a 25 to 40-fold increase of inhibitory activity respect to those of b2 and d1, respectively, 2-fold from Alogliptin (IC50 = 4 nM), and remarkable selectivity over DPP-8 and DPP-9 (>2000 fold). Further docking studies confirmed that the pyrazolo [1,5-a]pyrimidine core interacts with the S1 pocket whereas its substituted aromatic ring interacts with the sub-S1 pocket. The interactive mode in this case resembles that of Alogliptin and Trelagliptin. Further in vivo IPGTT assays in diabetic mice demonstrated that c24 effectively reduces glucose excursion by 48% at the dose of 10 mg/kg, suggesting that c24 is worthy of further development as a potent anti-diabetes agent.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/uso terapéutico , Hipoglucemiantes/uso terapéutico , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Inhibidores de la Dipeptidil-Peptidasa IV/síntesis química , Inhibidores de la Dipeptidil-Peptidasa IV/metabolismo , Diseño de Fármacos , Células Hep G2 , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/metabolismo , Masculino , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Relación Estructura-ActividadRESUMEN
Herein a series of Geniposide derivatives were designed, synthesized and evaluated as protein tyrosine phosphatase 1B (PTPlB) inhibitors. Most of these compounds exhibited potent in vitro PTP1B inhibitory activities, the representative 7a and 17f were found to be the most potent inhibitors against the enzyme with IC50 values of 0.35 and 0.41 µM, respectively. More importantly, they showcased 4 to10-fold selectivity over SHP2 and 3-fold over TCPTP. Further biological activity studies revealed that compounds 7a, 17b and 17f could effectively enhance insulin-stimulated glucose uptake with no significant cytotoxicity. Subsequent molecular docking and structural activity relationship analyses demonstrated that the glucose scaffold, benzylated glycosyl groups, and arylethenesulfonic acid ester significantly impact on the activity and selectivity.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Iridoides/síntesis química , Iridoides/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Transporte Biológico/efectos de los fármacos , Línea Celular , Técnicas de Química Sintética , Inhibidores Enzimáticos/química , Glucosa/metabolismo , Concentración 50 Inhibidora , Insulina/farmacología , Iridoides/química , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Ácidos Sulfónicos/químicaRESUMEN
Heavy metals, such as lead, mercury, and selenium, have been epidemiologically linked with a risk of ALS, but a molecular mechanism proving the connection has not been shown. A screen of putative developmental neurotoxins demonstrated that heavy metals (lead, mercury, and tin) trigger accumulation of TDP-43 into nuclear granules with concomitant loss of diffuse nuclear TDP-43. Lead (Pb) and methyl mercury (MeHg) disrupt the homeostasis of TDP-43 in neurons, resulting in increased levels of transcript and increased splicing activity of TDP-43. TDP-43 homeostasis is tightly regulated, and positively or negatively altering its splicing-suppressive activity has been shown to be deleterious to neurons. These changes are associated with the liquid-liquid phase separation of TDP-43 into nuclear bodies. We show that lead directly facilitates phase separation of TDP-43 in a dose-dependent manner in vitro, possibly explaining the means by which lead treatment results in neuronal nuclear granules. Metal toxicants also triggered the accumulation of insoluble TDP-43 in cultured cells and in the cortices of exposed mice. These results provide novel evidence of a direct mechanistic link between heavy metals, which are a commonly cited environmental risk of ALS, and molecular changes in TDP-43, the primary pathological protein accumulating in ALS.