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The opioid receptors play a pivotal role in the treatment of several neuropsychiatric and neurological disorders. Oroxylum indicum (L.) Kurtz is a very important medicinal plant with several therapeutic applications. It is a main constituent of the Ayurvedic formulation 'Dashmool' used for multifaceted disorders by the Indians. However, the constituents of this plant in neurological conditions have not been well studied. Here, we performed activity-guided isolation of compounds for opioid receptor modulator activity. In the study, we found that the isolated compound baicalein (3) has shown the most potent and competitive antagonistic activity at 20 mg/kg dose in vivo experiments. The acute dose of 3 (20 mg/kg) and pan opioid receptor antagonist naloxone (20 mg/kg) block the morphine-induced antinociception and the paw withdrawal latency decreases up to 8.3 s and 9.6 s, respectively. The in silico studies also support our in vitro data that compound 3 binds with MOR and KOR.
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The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, discover potential non-canonical signaling pathways, and determine the cryoelectron microscopy (cryo-EM) structure in complex with the chemokine CCL7. The structure reveals a distinct binding mode of chemokines, as reflected by relatively superficial binding and a partially formed orthosteric binding pocket. We also observe a dramatic shortening of TM5 and 6 on the intracellular side, which precludes the formation of the docking site for canonical signal transducers, thereby providing a possible explanation for the distinct pharmacological and functional phenotype of this receptor.
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Microscopia Crioeletrônica , Sistema do Grupo Sanguíneo Duffy , Receptores de Superfície Celular , Humanos , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/química , Sistema do Grupo Sanguíneo Duffy/metabolismo , Sistema do Grupo Sanguíneo Duffy/química , Transdução de Sinais , Sítios de Ligação , Quimiocinas/metabolismo , Quimiocinas/química , Ligação ProteicaRESUMO
Treatment-resistant depression (TRD) occurs in almost 50% of the depressed patients. Central kappa opioid receptor (KOR) agonism has been demonstrated to induce depression and anxiety, while KOR antagonism alleviates depression-like symptoms in rodent models and TRD in clinical studies. Previously, we have shown that sustained KOR activation leads to a TRD-like phenotype in mice, and modulation of brain-derived neurotrophic factor (BDNF) expression in the prefrontal cortex (PFC) appears to be one of the molecular determinants of the antidepressant response. In the present study, we observed that sustained KOR activation by a selective agonist, U50488, selectively reduced the levels of Bdnf transcripts II, IV, and Bdnf CDS (protein-coding Exon IX) in the PFC and cultured primary cortical neurons, which was blocked by selective KOR antagonist, norbinaltorphimine. Considering the crucial role of epigenetic pathways in BDNF expression, we further investigated the role of various epigenetic markers in KOR-induced BDNF downregulation in mice. We observed that treatment with U50488 resulted in selective and specific downregulation of acetylation at the ninth lysine residue of the histone H3 protein (H3K9ac) and upregulation of histone deacetylase 5 (HDAC5) expression in the PFC. Further, using anti-H3K9ac and anti-HDAC5 antibodies in the chromatin immune precipitation assay, we detected decreased enrichment of H3K9ac and increased HDAC5 binding at Bdnf II and IV transcripts after U50488 treatment, which were blocked by a selective KOR antagonist, norbinaltorphimine. Further mechanistic studies using HDAC5 selective inhibitor, LMK235, in primary cortical neurons and adeno-associated viral shRNA-mediated HDAC5-knockdown in the PFC of mice demonstrated an essential role of HDAC5 in KOR-mediated reduction of Bdnf expression in the PFC and in depression-like symptoms in mice. These results suggest that KOR engages multiple pathways to induce depression-like symptoms in mice and provide novel insights into the mechanisms by which activation of KOR regulates major depressive disorders.
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PROBLEM: Lipopolysaccharide (LPS) from gram-negative bacteria has reportedly been associated with infectious diseases like metritis, which has a substantial adverse effect on animal reproductive performance and causes serious financial losses for the dairy sector. The current work aimed to establish the impact of LPS on in vitro oocyte maturation and subsequent in vitro developmental competence of oocytes, as well as to investigate the explanatory molecular mechanism underlying this effect. METHOD OF STUDY: Buffalo cumulus-oocyte complexes (COCs) were challenged with 0, 5, 10 and 20 µg/mL LPS during IVM followed by IVF and IVC. Cytoplasmic and nuclear maturation, cleavage and blastocyst rate, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP, ΔΨm) and transcript abundance of genes related to inflammation, antioxidation and apoptosis were evaluated. RESULTS: The maturation and subsequent embryonic development competency were found to be significantly (p ≤ 0.05) reduced with the addition of 10 and 20 µg/mL LPS to IVM media. ROS production accompanied by a decreased ΔΨm was recorded in LPS-treated oocytes in comparison to the control group (p ≤ 0.05). Our results were further supported by the transcriptional expression of proinflammatory (TLR4, CD14 and RPS27A) and apoptotic gene (Caspase 3) which were found to be significantly increased while antioxidant genes (SOD2 and GPX1) were decreased significantly in matured oocytes and blastocyst after LPS exposure. CONCLUSIONS: The deleterious effects of LPS are mediated through ROS generation, which triggers inflammatory processes via the TLR4 pathway and impairs oocyte maturation and subsequent embryonic development.
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Búfalos , Desenvolvimento Embrionário , Técnicas de Maturação in Vitro de Oócitos , Lipopolissacarídeos , Mitocôndrias , Oócitos , Espécies Reativas de Oxigênio , Transdução de Sinais , Receptor 4 Toll-Like , Animais , Receptor 4 Toll-Like/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oócitos/metabolismo , Oócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células Cultivadas , Blastocisto/metabolismo , Blastocisto/efeitos dos fármacos , Fertilização in vitroRESUMO
Genome editing has enhanced our ability to understand the role of genetics in a number of diseases by facilitating the development of more precise cellular and animal models to study pathophysiological processes. These advances have shown extraordinary promise in a multitude of areas, from basic research to applied bioengineering and biomedical research. Induced pluripotent stem cells (iPSCs) are known for their high replicative capacity and are excellent targets for genetic manipulation as they can be clonally expanded from a single cell without compromising their pluripotency. Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR/Cas RNA-guided nucleases have rapidly become the method of choice for gene editing due to their high specificity, simplicity, low cost, and versatility. Coupling the cellular versatility of iPSCs differentiation with CRISPR/Cas9-mediated genome editing technology can be an effective experimental technique for providing new insights into the therapeutic use of this technology. However, before using these techniques for gene therapy, their therapeutic safety and efficacy following models need to be assessed. In this review, we cover the remarkable progress that has been made in the use of genome editing tools in iPSCs, their applications in disease research and gene therapy as well as the hurdles that remain in the actual implementation of CRISPR/Cas systems.
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Edição de Genes , Células-Tronco Pluripotentes Induzidas , Animais , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Terapia Genética/métodos , Diferenciação CelularRESUMO
X-linked hypophosphatemia (XLH) is the most common form of hereditary hypophosphatemic rickets. The genetic basis for XLH is loss of function mutations in the phosphate-regulating endopeptidase X-linked (PHEX), which leads to increased circulating fibroblast growth factor 23 (FGF23). This increase in FGF23 impairs activation of vitamin D and attenuates renal phosphate reabsorption, leading to rickets. Previous studies have demonstrated that ablating FGF23 in the Hyp mouse model of XLH leads to hyperphosphatemia, high levels of 1,25-dihydroxyvitamin D, and is not associated with the development of rickets. Studies were undertaken to define a role for the increase in 1,25-dihydroxyvitamin D levels in the prevention of rickets in Hyp mice lacking FGF23. These mice were mated to mice lacking Cyp27b1, the enzyme responsible for activating vitamin D metabolites, to generate Hyp mice lacking both FGF23 and 1,25-dihydroxyvitamin D (FCH mice). Mice were fed a special diet to maintain normal mineral ion homeostasis. Despite normal mineral ions, Hyp mice lacking both FGF23 and Cyp27b1 developed rickets, characterized by an interrupted, expanded hypertrophic chondrocyte layer and impaired hypertrophic chondrocyte apoptosis. This phenotype was prevented when mice were treated with 1,25-dihydroxyvitamin D from day 2 until sacrifice on day 30. Interestingly, mice lacking FGF23 and Cyp27b1 without the PHEX mutation did not exhibit rickets. These findings define an essential PHEX-dependent, FGF23-independent role for 1,25-dihydroxyvitamin D in XLH and have important therapeutic implications for the treatment of this genetic disorder.
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Raquitismo Hipofosfatêmico Familiar , Animais , Camundongos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Raquitismo Hipofosfatêmico Familiar/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Lâmina de Crescimento/metabolismo , Minerais/uso terapêutico , Fosfatos , Vitamina D/metabolismoRESUMO
Electroporation is a widely used method for delivering CRISPR components into cells; however, it presents challenges when applied to difficult-to-transfect cells like adult buffalo fibroblasts. In this study, the ITGB2 gene (encoding the CD18 protein), plays vital for cellular adhesion and immune responses, was selected for editing experiments. To optimize electroporation conditions, we investigated parameters such as electric field strength, pulse duration, plasmid DNA amount, cuvette type, and cell type. The best transfection rates were obtained in a 4 mm gap cuvette with a single 20-millisecond pulse of 300 V using a 10 µg of all-in-one CRISPR plasmid for 106 cells in 100 µL of electroporation buffer. Increasing DNA quantity enhanced transfection rates but compromised cell viability. The 4 mm cuvette gap had high transfection rates than the 2 mm gap, and newborn cells exhibited higher transfection rates than adult cells. We achieved transfection rates of 10-12% with a cell viability of 25-30% for adult fibroblast cells. Subsequently, successfully edited the ITGB2 gene with a 30% editing efficiency, confirmed through various analysis methods, including T7E1 assay, TIDE and ICE analysis, and TA cloning. In conclusion, electroporation conditions reported here can edit buffalo gene(s) for various biotechnological research applications.
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Búfalos , Edição de Genes , Animais , Edição de Genes/métodos , Búfalos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Eletroporação , Transfecção , Fibroblastos , DNA , Sistemas CRISPR-Cas/genéticaRESUMO
Solvent shielding of the amide hydrogen bond donor (NH groups) through chemical modification or conformational control has been successfully utilized to impart membrane permeability to macrocyclic peptides. We demonstrate that passive membrane permeability can also be conferred by masking the amide hydrogen bond acceptor (>C = O) through a thioamide substitution (>C = S). The membrane permeability is a consequence of the lower desolvation penalty of the macrocycle resulting from a concerted effect of conformational restriction, local desolvation of the thioamide bond, and solvent shielding of the amide NH groups. The enhanced permeability and metabolic stability on thioamidation improve the bioavailability of a macrocyclic peptide composed of hydrophobic amino acids when administered through the oral route in rats. Thioamidation of a bioactive macrocyclic peptide composed of polar amino acids results in analogs with longer duration of action in rats when delivered subcutaneously. These results highlight the potential of O to S substitution as a stable backbone modification in improving the pharmacological properties of peptide macrocycles.
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Amidas , Tioamidas , Ratos , Animais , Amidas/química , Tioamidas/química , Disponibilidade Biológica , Peptídeos , Permeabilidade , Aminoácidos , SolventesRESUMO
Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore multiple aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, in terms of their transducer-coupling and downstream signaling upon their stimulation by a common chemokine agonist, CXCL12, and a small molecule agonist, VUF11207. We observe that CXCR7 lacks G-protein-coupling while maintaining robust ßarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected but exhibits significantly reduced ßarr-coupling compared to CXCR7. These two receptors induce distinct ßarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We also identify a key contribution of a single phosphorylation site in CXCR7 for ßarr recruitment and endosomal localization. Our study provides molecular insights into intrinsic-bias encoded in the CXCR4-CXCR7 system with broad implications for drug discovery.
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Receptores CXCR , Receptores CXCR/genética , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Proteínas de Ligação ao GTP , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Quimiocina CXCL12/metabolismoRESUMO
Low circulating phosphate (Pi) leads to rickets, characterized by expansion of the hypertrophic chondrocytes (HCs) in the growth plate due to impaired HC apoptosis. Studies in HCs demonstrate that Pi activates the Raf/MEK/ERK1/2 and mitochondrial apoptotic pathways. To determine how Pi activates these pathways, a small-molecule screen was undertaken to identify inhibitors of Pi-induced ERK1/2 phosphorylation in HCs. Vascular endothelial growth factor receptor 2 (VEGFR2) was identified as a target. In vitro studies in HCs demonstrate that VEGFR2 inhibitors block Pi-induced pERK1/2 and caspase-9 cleavage. Like Pi, rhVEGF activates ERK1/2 and caspase-9 in HCs and induces phosphorylation of VEGFR2, confirming that Pi activates this signaling pathway in HCs. Chondrocyte-specific depletion of VEGFR2 leads to an increase in HCs, impaired vascular invasion, and a decrease in HC apoptosis. Thus, these studies define a role for VEGFR2 in transducing Pi signals and mediating its effects on growth plate maturation.
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Despite ongoing advancements in research, the inability of therapeutics to cross the blood-brain barrier (BBB) makes the treatment of neurological disorders (NDs) a challenging task, offering only partial symptomatic relief. Various adverse effects associated with existing approaches are another significant barrier that prompts the usage of structurally diverse phytochemicals as preventive/therapeutic lead against NDs in preclinical and clinical settings. Despite numerous beneficial properties, phytochemicals suffer from poor pharmacokinetic profile which limits their pharmacological activity and necessitates the utility of nanotechnology for efficient drug delivery. Nanocarriers have been shown to be proficient carriers that can enhance drug delivery, bioavailability, biocompatibility, and stability of phytochemicals. We, thus, conducted a meticulous literature survey using several electronic databases to gather relevant studies in order to provide a comprehensive summary about the use of nanocarriers in delivering phytochemicals as a treatment approach for NDs. Additionally, the review highlights the mechanisms of drug transport of nanocarriers across the BBB and explores their potential future applications in this emerging field.
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Portadores de Fármacos , Nanopartículas , Portadores de Fármacos/química , Nanopartículas/química , Encéfalo , Barreira Hematoencefálica , Sistemas de Liberação de Medicamentos , Compostos Fitoquímicos/uso terapêutico , Compostos Fitoquímicos/farmacologiaRESUMO
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and somatic cell nuclear transfer (SCNT) have been used to produce genome-edited farm animal species for improved production and health traits; however, these tools are rarely used in the buffalo and can play a pivotal role in milk and meat production in tropical and subtropical countries. In this study, we aimed to produce myostatin (MSTN) gene-edited embryos of the Murrah buffalo using the CRISPR/Cas9 system and SCNT. For this, fibroblast cells were electroporated with sgRNAs carrying all-in-one CRISPR/Cas9 plasmids targeting the first exon of the MSTN gene. Following puromycin selection, single-cell clonal populations were established and screened using the TA cloning and Sanger sequencing methods. Of eight single-cell clonal populations, one with a monoallelic and another with a biallelic heterozygous gene editing event were identified. These two gene-edited clonal cell populations were successfully used to produce blastocyst-stage embryos using the handmade cloning method. This work establishes the technical foundation for generation of genome-edited cloned embryos in the buffalo.
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Sistemas CRISPR-Cas , Edição de Genes , Animais , Edição de Genes/métodos , Técnicas de Transferência Nuclear/veterinária , Clonagem de Organismos , BlastocistoRESUMO
The emergence and rapid evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a global crisis that required a detailed characterization of the dynamics of mutational pattern of the viral genome for comprehending its epidemiology, pathogenesis and containment. We investigated the molecular evolution of the SASR-CoV-2 genome during the first, second and third waves of COVID-19 in Uttar Pradesh, India. Nanopore sequencing of the SARS-CoV-2 genome was undertaken in 544 confirmed cases of COVID-19, which included vaccinated and unvaccinated individuals. In the first wave (unvaccinated population), the 20A clade (56.32%) was superior that was replaced by 21A Delta in the second wave, which was more often seen in vaccinated individuals in comparison to unvaccinated (75.84% versus 16.17%, respectively). Subsequently, 21A delta got outcompeted by Omicron (71.8%), especially the 21L variant, in the third wave. We noticed that Q677H appeared in 20A Alpha and stayed up to Delta, D614G appeared in 20A Alpha and stayed in Delta and Omicron variants (got fixed), and several other mutations appeared in Delta and stayed in Omicron. A cross-sectional analysis of the vaccinated and unvaccinated individuals during the second wave revealed signature combinations of E156G, F157Del, L452R, T478K, D614G mutations in the Spike protein that might have facilitated vaccination breach in India. Interestingly, some of these mutation combinations were carried forward from Delta to Omicron. In silico protein docking showed that Omicron had a higher binding affinity with the host ACE2 receptor, resulting in enhanced infectivity of Omicron over the Delta variant. This work has identified the combinations of key mutations causing vaccination breach in India and provided insights into the change of [virus's] binding affinity with evolution, resulting in more virulence in Delta and more infectivity in Omicron variants of SARS-CoV-2. Our findings will help in understanding the COVID-19 disease biology and guide further surveillance of the SARS-CoV-2 genome to facilitate the development of vaccines with better efficacies.
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Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for the introduction of mutations using CRISPR-Cas9 electroporation of zygote (CRISPR-EP) in buffalo. The goal of the study was to determine the optimal conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced buffalo zygotes by electroporation. Electroporation was performed using different combinations of voltage, pulse and time, and we observed that the electroporation in buffalo zygote at 20 V/mm, 5 pulses, 3 msec at 10 h post insemination (hpi) resulted in increased membrane permeability and higher knockout efficiency without altering embryonic developmental potential. Using the above parameters, we targeted buffalo POU5F1 gene as a proof of concept and found no variations in embryonic developmental competence at cleavage or blastocyst formation rate between control, POU5F1-KO, and electroporated control (EC) embryos. To elucidate the effect of POU5F1-KO on other pluripotent genes, we determined the relative expression of SOX2, NANOG, and GATA2 in the control (POU5F1 intact) and POU5F1-KO-confirmed blastocyst. POU5F1-KO significantly (p ≤ 0.05) altered the expression of SOX2, NANOG, and GATA2 in blastocyst stage embryos. In conclusion, we standardized an easy and straightforward protocol CRISPR-EP method that could be served as a useful method for studying the functional genomics of buffalo embryos.
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Pain sensation is a normal physiological response to alert and prevent further tissue damage. It involves the perception of external stimuli by somatosensory neurons, then transmission of the message to various other types of neurons present in the spinal cord and brain to generate an appropriate response. Currently available analgesics exhibit very modest efficacy, and that too in only a subset of patients with chronic pain conditions, particularly neuropathic pain. The G protein-coupled receptors (GPCRs) are expressed on presynaptic, postsynaptic terminals, and soma of somatosensory neurons, which binds to various types of ligands to modulate neuronal activity and thus pain sensation in both directions. Fundamentally, neuropathic pain arises due to aberrant neuronal plasticity, which includes the sensitization of peripheral primary afferents (dorsal root ganglia and trigeminal ganglia) and the sensitization of central nociceptive neurons in the spinal cord or trigeminal nucleus or brain stem and cortex. Owing to the expression profiles of GPCRs in somatosensory neurons and other neuroanatomical regions involved in pain processing and transmission, this article shall focus only on four families of GPCRs: 1- Opioid receptors, 2-Cannabinoid receptors, 3-Adenosine receptors, and 4-Chemokine receptors.
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Neuralgia , Humanos , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Gânglios Espinais/metabolismo , Medula Espinal/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Chlorophyll, the principal photoacceptor of green plants, plays a pivotal role in photosynthesis. In the recent past, chlorophyll has also been utilized as an efficient organo-photocatalyst in several organic syntheses. The inexpensive, ubiquitous nature of chlorophyll endorses it as an appealing green alternative to transition metal photocatalysts. This review is the first attempt to showcase and analyze the photocatalytic activity of chlorophyll in effecting different organic transformations. We intend to provide a holistic overview of the role of chlorophyll starting from photosynthesis to its contemporary synthetic applications in visible light photocatalysis. In addition, the photophysical and electrochemical properties of chlorophyll are elaborated to attain a clearer understanding of its mode of action as a visible light photocatalyst.
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Clorofila , Elementos de Transição , Catálise , Luz , Elementos de Transição/química , Técnicas de Química SintéticaRESUMO
Efforts have been devoted for the discovery and development of positive allosteric modulators (PAMs) of 5-HT2CR because of their potential advantages over the orthosteric agonist like Lorcaserin that was withdrawn from the market. On the other hand, pursuing a positive ago-allosteric modulator (PAAM) is considered as beneficial particularly when an agonist is not capable of affecting the potency of the endogenous agonist sufficiently. In search of a suitable PAAM of 5-HT2CR we adopted an in silico based approach that indicated the potential of the 3-(1-hydroxycycloalkyl) substituted isoquinolin-1-one derivatives against the 5-HT2CR as majority of these molecules interacted with the site other than that of Lorcaserin with superior docking scores. These compounds along with the regioisomeric 3-methyleneisoindolin-1-one derivatives were prepared via the Cu(OAc)2 catalyzed coupling of 2-iodobenzamide with 1-ethynylcycloalkanol under ultrasound irradiation. According to the in vitro studies, most of these compounds were not only found to be potent and selective agonists but also emerged as PAAM of 5-HT2CR whereas Lorcaserin did not show PAAM activities. According to the SAR study the isoquinolin-1(2H)-ones appeared as better PAAM than isoindolin-1-ones whereas the presence of hydroxyl group appeared to be crucial for the activity. With the potent PAAM activity for 5-HT2CR (EC50 = 1 nM) and 107 and 86-fold selectivity towards 5-HT2C over 5-HT2A and 5-HT2B the compound 4i was identified as a hit molecule. The compound showed good stability in male BALB/c mice brain homogenate (â¼85 % remaining after 2 h), moderate stability in the presence of rat liver microsomes (42 % remaining after 1 h) and acceptable PK properties with fast reaching in the brain maintaining â¼ 1:1 brain/plasma concentration ratio. The compound at a dose of 50 mg/kg exhibited decreased trend in the food intake starting from day 3 in S.D. rats, which reached significant by 5th day, and the effect was comparable to Lorcaserin (10 mg/kg) on day 5. Thus, being the first example of PAAM of 5-HT2CR the compound 4i is of further medicinal interest.
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Indóis , Isoquinolinas , Agonistas do Receptor 5-HT2 de Serotonina , Animais , Masculino , Camundongos , Ratos , Encéfalo , Agonistas do Receptor 5-HT2 de Serotonina/síntese química , Agonistas do Receptor 5-HT2 de Serotonina/química , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Camundongos Endogâmicos BALB C , Isoquinolinas/síntese química , Isoquinolinas/química , Isoquinolinas/farmacologia , Indóis/síntese química , Indóis/química , Indóis/farmacologiaRESUMO
Pyrazolo[1,5-a]quinoxalin-4(5H)-one derivatives as novel opioid receptor modulators have been synthesized via copper-catalyzed oxidative [3 + 2]-annulation of quinoxalin-2(1H)-one and oxime-O-acetates. This hydrazine-free C-C and N-N bond formation strategy starts with the generation of C2N1 synthon using oxime acetate, which reacts in a [3 + 2] manner with quinoxalin-2(1H)-one, followed by oxidative aromatization. The synthesized compounds were tested against opioid receptors, of which eight compounds exhibited an antagonistic effect with EC50 < 5 µM at various opioid receptors. Molecular docking studies were performed to identify the binding of active pyrazolo[1,5-a]quinoxalin-4(5H)-one ligands with hKOR protein. Docking results indicated that compounds 3d and 3g participate in hydrogen bonding with the hydroxyl group of T111 of the active site pocket residue.
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Oximas , Quinoxalinas , Catálise , Cobre , Ésteres , Simulação de Acoplamento Molecular , Estresse Oxidativo , Oximas/farmacologia , Quinoxalinas/química , Quinoxalinas/farmacologia , Receptores OpioidesRESUMO
Sperm mitochondrion is one of the major susceptible organelles that get damaged during cryopreservation. The study aimed to minimize mitochondrial dysfunction and oxidative stress during sperm cryopreservation using mitochondria-specific antioxidants. For this, semen was collected from five buffalo bulls (3 ejaculates/bull). The ejaculates were diluted in an low-density lipoprotein-based extender and divided into four equal aliquots. Mitochondria-targeted antioxidant (MitoQ) was added at a final concentration of 0 (control), 0.02, 0.2 and 2 µM separately in each aliquotes and cryopreserved. The addition of MitoQ at a concentration of 0.02 µM improved post-thaw sperm motility, plasma membrane integrity and able to sustain sperm motility for a longer time. To investigate MitoQ's effects on mitochondrial function, we measured mitochondrial membrane potential (MMP) using JC-1 dye, superoxide production using Mitosox assay, and lipid peroxidation by TBARS assay. The supplementation of 0.02 µM MitoQ in the extender prevented the significant reduction of MMP and reduced superoxide production resulting in lower lipid peroxidation of sperm plasma membrane after cryopreservation. Further, we found that a higher concentration of MitoQ decreases MMP and increases mitochondrial superoxide production. In conclusion, MitoQ @ 0.02 µM can alleviate oxidative stress by regulating mitochondrial functionality in spermatozoa during cryopreservation.
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Antioxidantes , Preservação do Sêmen , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Búfalos/fisiologia , Criopreservação/métodos , Crioprotetores/farmacologia , Masculino , Mitocôndrias/metabolismo , Análise do Sêmen , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides , Espermatozoides , Superóxidos/metabolismoRESUMO
Photocatalytic syntheses of quinoline-2(1H)-ones, isoquinoline-1(2H)-ones and 1,2,4-trioxanes were achieved by selective photo-induced electron transfer (PET) and energy transfer (EnT), respectively, by chlorophyll under visible light irradiation. Quinoline-2(1H)-ones, isoquinoline-1(2H)-ones and 1,2,4-trioxanes are biologically potent scaffolds and their syntheses following mild reaction protocols are highly sought after. This work showcases the divergent photocatalytic roles of chlorophyll viz., electron transfer in the case of quinolines or isoquinolines and energy transfer with allyl alcohols as substrates, affording their aerobic oxidation under green reaction conditions. The mechanistic investigations affirm that the catalytic cycle follows the electron-transfer pathway in carrying out the oxidation of N-alkyl(iso)quinolinium salts. Furthermore, the method provides an environmentally benign, simple reaction strategy for organic transformations of (N)-heterocycles.