RESUMO
Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch22.1Ecan mice, and following the expression of the NOTCH2 intracellular domain in vitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes.
Assuntos
Condrócitos , Osteoartrite , Receptor Notch2 , Fator de Necrose Tumoral alfa , Animais , Humanos , Camundongos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Imunoglobulinas , Interleucina-6/genética , Interleucina-6/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Inflamação , Modelos Animais de Doenças , Condrogênese , Transdução de Sinais/efeitos dos fármacos , Domínios Proteicos/imunologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
The integrated dysbiosis of gut microbiota and altered host transcriptomics in irritable bowel syndrome (IBS) is yet to be known. This study investigated the associations among gut microbiota and host transcriptomics in young adults with IBS. Stool and peripheral blood samples from 20 IBS subjects and 21 healthy controls (HCs) collected at the baseline visit of an RCT were sequenced to depict the gut microbiota and transcriptomic profiles, respectively. The diversities, composition, and predicted metabolic pathways of gut microbiota significantly differed between IBS subjects and HCs. Nine genera were significantly abundant in IBS stool samples, including Akkermansia, Blautia, Coprococcus, Granulicatella, Holdemania, Oribacterium, Oscillospira, Parabacteroides, and Sutterella. There were 2264 DEGs found between IBS subjects and HCs; 768 were upregulated, and 1496 were downregulated in IBS participants compared with HCs. The enriched gene ontology included the immune system process and immune response. The pathway of antigen processing and presentation (hsa04612) in gut microbiota was also significantly different in the RNA-seq data. Akkermansia, Blautia, Holdemania, and Sutterella were significantly correlated with ANXA2P2 (upregulated, positive correlations), PCSK1N (downregulated, negative correlations), and GLTPD2 (downregulated, negative correlations). This study identified the dysregulated immune response and metabolism in IBS participants revealed by the altered gut microbiota and transcriptomic profiles.
Assuntos
Microbioma Gastrointestinal , Síndrome do Intestino Irritável , Humanos , Adulto Jovem , Síndrome do Intestino Irritável/metabolismo , Multiômica , Microbioma Gastrointestinal/fisiologia , Fezes/microbiologia , Firmicutes/genética , Imunidade , Perfilação da Expressão GênicaRESUMO
A new paradigm has slowly emerged regarding the diversification of bryophytes, with inferences from molecular data highlighting a dynamic evolution of their genome. However, comparative studies of expressed genes among closely related taxa is so far missing. Here we contrast the dimensions of the vegetative transcriptome of Funaria hygrometrica and Physcomitrium pyriforme against the genome of their relative, Physcomitrium (Physcomitrella) patens. These three species of Funariaceae share highly conserved vegetative bodies, and are partially sympatric, growing on mineral soil in mostly temperate regions. We analyzed the vegetative gametophytic transcriptome of F. hygrometrica and P. pyriforme and mapped short reads, transcripts, and proteins to the genome and gene space of P. patens. Only about half of the transcripts of F. hygrometrica map to their ortholog in P. patens, whereas at least 90% of those of P. pyriforme align to loci in P. patens. Such divergence is unexpected given the high morphological similarity of the gametophyte but reflects the estimated times of divergence of F. hygrometrica and P. pyriforme from P. patens, namely 55 and 20 mya, respectively. The newly sampled transcriptomes bear signatures of at least one, rather ancient, whole genome duplication (WGD), which may be shared with one reported for P. patens. The transcriptomes of F. hygrometrica and P. pyriforme reveal significant contractions or expansions of different gene families. While transcriptomes offer only an incomplete estimate of the gene space, the high number of transcripts obtained suggest a significant divergence in gene sequences, and gene number among the three species, indicative of a rather strong, dynamic genome evolution, shaped in part by whole, partial or localized genome duplication. The gene ontology of their specific and rapidly-evolving protein families, suggests that the evolution of the Funariaceae may have been driven by the diversification of metabolic genes that may optimize the adaptations to environmental conditions, a hypothesis well in line with ecological patterns in the genetic diversity and structure in seed plants.
Assuntos
Bryopsida/anatomia & histologia , Bryopsida/genética , Filogenia , Evolução Molecular , Genoma de Planta , Família Multigênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genéticaRESUMO
BACKGROUND: Adolescent idiopathic scoliosis is one of the most common spinal deformities in children and adolescents requiring extensive surgical intervention. Due to the nature of surgery, spinal fusion increases their risk of experiencing persistent postsurgical pain. Up to 20% of adolescents report pain for months or years after corrective spinal fusion surgery. AIMS: To examine the influence of preoperative psychosocial factors and mRNA expression profiles on persistent postoperative pain in adolescents undergoing corrective spinal fusion surgery. DESIGN: Prospective, longitudinal cohort study. SETTING: Two freestanding academic children's hospitals. METHODS: Utilizing a longitudinal approach, adolescents were evaluated at baseline (preoperatively) and postoperatively at ±1 month and ±4-6 months. Self-report of pain scores, the Pain Catastrophizing Scale-Child, and whole blood for RNA sequencing analysis were obtained at each time point. RESULTS: Of the adolescents enrolled in the study, 36% experienced persistent pain at final postoperative follow-up. The most significant predictors of persistent pain included preoperative pain severity and helplessness. Gene expression analysis identified HLA-DRB3 as having increased expression in children who experienced persistent pain postoperatively, as opposed to those whose pain resolved. A prospective validation study with a larger sample size is needed to confirm these findings. CONCLUSIONS: While adolescent idiopathic scoliosis is not often classified as a painful condition, providers must be cognizant of pre-existing pain and anxiety that may precipitate a negative recovery trajectory. Policy and practice change are essential for early identification and subsequent intervention.
Assuntos
Catastrofização , Fusão Vertebral , Adolescente , Criança , Expressão Gênica , Humanos , Estudos Longitudinais , Dor Pós-Operatória , Fusão Vertebral/efeitos adversosRESUMO
Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.
Assuntos
Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Histonas/química , Histonas/metabolismo , Acetilação , Sítios de Ligação , Replicação do DNA , Chaperonas de Histonas/genética , Histonas/genética , Modelos Biológicos , Nucleossomos/metabolismoRESUMO
Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase's most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements.
Assuntos
Adenosina Trifosfatases/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Nucleossomos/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Transcrição Gênica , Adenosina Trifosfatases/metabolismo , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila , Regulação da Expressão Gênica , Células HeLa , Humanos , Nucleossomos/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%-40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at â²5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.
Assuntos
Análise de Sequência de RNA/métodos , Perfilação da Expressão Gênica , RNA Fúngico/genética , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Genomes of eukaryotes exist as chromatin, and it is known that different chromatin states can influence gene regulation. Chromatin is not a static structure, but is known to be dynamic and vary between cells. In order to monitor the organisation of chromatin in live cells we have engineered fluorescent fusion proteins which recognize specific operator sequences to tag pairs of syntenic gene loci. The separation of these loci was then tracked in three dimensions over time using fluorescence microscopy. RESULTS: We established a work flow for measuring the distance between two fluorescently tagged, syntenic gene loci with a mean measurement error of 63 nm. In general, physical separation was observed to increase with increasing genomic separations. However, the extent to which chromatin is compressed varies for different genomic regions. No correlation was observed between compaction and the distribution of chromatin markers from genomic datasets or with contacts identified using capture based approaches. Variation in spatial separation was also observed within cells over time and between cells. Differences in the conformation of individual loci can persist for minutes in individual cells. Separation of reporter loci was found to be similar in related and unrelated daughter cell pairs. CONCLUSIONS: The directly observed physical separation of reporter loci in live cells is highly dynamic both over time and from cell to cell. However, consistent differences in separation are observed over some chromosomal regions that do not correlate with factors known to influence chromatin states. We conclude that as yet unidentified parameters influence chromatin configuration. We also find that while heterogeneity in chromatin states can be maintained for minutes between cells, it is not inherited through cell division. This may contribute to cell-to-cell transcriptional heterogeneity.
Assuntos
Divisão Celular , Cromatina/metabolismo , Imageamento Tridimensional/métodos , Anisotropia , Sobrevivência Celular , Genes Reporter , Genoma , Movimento (Física) , Dinâmica não Linear , Análise de Célula ÚnicaRESUMO
MOTIVATION: High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. RESULTS: A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of â¼0.01. The high-replicate data also allowed for strict quality control and screening of 'bad' replicates, which can drastically affect the gene read-count distribution. AVAILABILITY AND IMPLEMENTATION: RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. CONTACT: g.j.barton@dundee.ac.uk.
Assuntos
Modelos Estatísticos , Análise de Sequência de RNA/métodos , Sequência de Bases , Distribuição Binomial , Perfilação da Expressão Gênica , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genéticaRESUMO
Zingiber officinale Roscoe, commonly known as ginger, is a traditional herb used to treat various disorders. In this study, we evaluated potential pharmacological effects of ethanolic extracts of Z. Officinale with respect to central nervous system (CNS) activity in mice. Role of ethanolic extract of ginger on CNS activity in mice was studied using models of elevated plus maze test, barbiturate-induced sleeping time, tail suspension test, hot-plate and tail-flick test. Ginger extract was administered to mice at single doses of 50 and 200 mg/kg, perorally while diazepam (1 mg/kg), morphine (5 mg/kg) and imipramine (30 mg/kg) intraperitoneally were used as standard drugs. The results showed that the ginger extract at all dose levels significantly exhibited anxiolytic activityincreased the sleeping latency but reduced the sleeping time. Tail suspension test showed that the extract at both the doses was able to induce a significant decrease in the immobility time, similar to imipramine, a recognized antidepressant drug. Tail-flick and hot-plate tests demonstrated antinociceptive property of ginger extract, similar to morphine, a recognized antinociceptive agent. Higher dose level (200 mg/kg) showed better protective effects. Phytochemical screening of ethanolic extract revealed the presence of various phytoconstituents such as phenolic compounds, flavonoids, tannins, anthocyanins, carbohydrates, glycosides, proteins, resins and volatile oils. The possible mechanism by which ginger exhibited the significant beneficial effects on various CNS models in mice could be attributed to its antioxidant potential.
Assuntos
Analgésicos/farmacologia , Ansiolíticos/farmacologia , Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Etanol/química , Extratos Vegetais/farmacologia , Solventes/química , Analgésicos/isolamento & purificação , Animais , Ansiolíticos/isolamento & purificação , Antidepressivos/isolamento & purificação , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Relação Dose-Resposta a Droga , Feminino , Zingiber officinale/química , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Atividade Motora/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Tempo de Reação/efeitos dos fármacos , Sono/efeitos dos fármacosRESUMO
The ATP-dependent chromatin-remodelling enzyme Chd1 is a 168-kDa protein consisting of a double chromodomain, Snf2-related ATPase domain, and a C-terminal DNA-binding domain. Here, we show the DNA-binding domain is required for Saccharomyces cerevisiae Chd1 to bind and remodel nucleosomes. The crystal structure of this domain reveals the presence of structural homology to SANT and SLIDE domains previously identified in ISWI remodelling enzymes. The presence of these domains in ISWI and Chd1 chromatin-remodelling enzymes may provide a means of efficiently harnessing the action of the Snf2-related ATPase domain for the purpose of nucleosome spacing and provide an explanation for partial redundancy between these proteins. Site directed mutagenesis was used to identify residues important for DNA binding and generate a model describing the interaction of this domain with DNA. Through inclusion of Chd1 sequences in homology searches SLIDE domains were identified in CHD6-9 proteins. Point mutations to conserved amino acids within the human CHD7 SLIDE domain have been identified in patients with CHARGE syndrome.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Síndrome CHARGE/genética , Montagem e Desmontagem da Cromatina/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Nucleossomos/metabolismo , Organismos Geneticamente Modificados , Filogenia , Mutação Puntual , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites.
Assuntos
Adenosina Trifosfatases/metabolismo , Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Sítios de Ligação , Segregação de Cromossomos , DNA Fúngico/genética , DNA Fúngico/metabolismo , Deleção de Genes , Ligação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.
Assuntos
Proteínas de Bactérias/metabolismo , Imunidade Inata , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Neuropeptídeos/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Tuberculose Pulmonar/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/microbiologia , Linhagem Celular Transformada , Células Cultivadas , Feminino , Humanos , Macrófagos/enzimologia , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/fisiologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/genética , Núcleosídeo-Difosfato Quinase/antagonistas & inibidores , Núcleosídeo-Difosfato Quinase/genética , Oligorribonucleotídeos Antissenso , Fagossomos/enzimologia , Fagossomos/ultraestrutura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tuberculose Pulmonar/enzimologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Virulência , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Four newer, cost effective and sensitive ion-pair complex methods were estimated for the determination of eflornithine hydrochloride drug in pharmaceutical formulation. In these methods eflornithine hydrochloride react with bromocresol green (buffer of pH 4), bromophenol blue (buffer pH 4.5), methyl orange (buffer of pH 5.5) and bromothymol blue (buffer of pH 5) respectively. The chloroform was used for extraction of ion-pair complexes. The measurement of complexes was done at 413, 416, 417 and 425 nm respectively. Under the described conditions the proposed methods are linear over the concentration range of 3-18, 4-16, 6-30 and 2-12 µg/ml and the coefficient of determination were >0.999 (n=6) with a relative standard deviation of <1% (n=6). The average recovery of the target compound is >100% with a limit of quantification (LOQ) of 20, 0.869, 2 and 4.167 µg/ml and the limit of detection (LOD) 6.6, 0.287, 0.66 and 1.375 µg/ml. The mechanism of the derivatization reaction is proposed and advantages of the proposed method are discussed.
Assuntos
Eflornitina/análise , Espectrofotometria/métodos , Estabilidade de Medicamentos , Eflornitina/química , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Limite de DetecçãoRESUMO
Mycobacterium tuberculosis (Mtb) is a phenomenally successful human pathogen having evolved mechanisms that allow it to survive within the hazardous environment of macrophages and establish long term, persistent infection in the host against the control of cell-mediated immunity. One such mechanism is mediated by the truncated hemoglobin, HbN, of Mtb that displays a potent O2-dependent nitric oxide dioxygenase activity and protects its host from the toxicity of macrophage-generated nitric oxide (NO). Here we demonstrate for the first time that HbN is post-translationally modified by glycosylation in Mtb and remains localized on the cell membrane and the cell wall. The glycan linkage in the HbN was identified as mannose. The elevated expression of HbN in Mtb and M. smegmatis facilitated their entry within the macrophages as compared with isogenic control cells, and mutation in the glycan linkage of HbN disrupted this effect. Additionally, HbN-expressing cells exhibited higher survival within the THP-1 and mouse peritoneal macrophages, simultaneously increasing the intracellular level of proinflammatory cytokines IL-6 and TNF-α and suppressing the expression of co-stimulatory surface markers CD80 and CD86. These results, thus, suggest the involvement of HbN in modulating the host-pathogen interactions and immune system of the host apart from protecting the bacilli from nitrosative stress inside the activated macrophages, consequently driving cells toward increased infectivity and intracellular survival.
Assuntos
Proteínas de Bactérias/imunologia , Espaço Intracelular/imunologia , Mycobacterium tuberculosis/imunologia , Hemoglobinas Truncadas/imunologia , Sequência de Aminoácidos , Animais , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-2/imunologia , Antígeno B7-2/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Glicosilação , Interações Hospedeiro-Patógeno/imunologia , Humanos , Espaço Intracelular/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/fisiologia , Processamento de Proteína Pós-Traducional/imunologia , Homologia de Sequência de Aminoácidos , Hemoglobinas Truncadas/genética , Hemoglobinas Truncadas/metabolismoRESUMO
Micelles of the star shaped block polymers "tetronics" were employed for the synthesis of gold (Au) nanoparticles (NPs) under the effect of pH and temperature variation. The presence of the diamine core in the tetronic macromolecule made its micelles highly pH responsive, thereby dramatically altering the physiochemical properties. Likewise, a high degree of hydration made the micelles temperature sensitive. UV-visible studies, transmission electron microscopy (TEM), gel electrophoresis, and structure optimization by energy minimization were applied to understand the physiochemical aspects of tetronic micelles and their further role in the synthesis of Au NPs. Synthesis of Au NPs was triggered by the surface cavities of the micelles and hence the NPs simultaneously adsorbed on the micelle surface. Low pH induced high hydration and temperature responsive well defined vesicular morphologies bearing Au NPs, while high pH produced mainly large and compact compound micelles carrying NPs. Both pH and temperature responsive behaviors of different tetronics significantly influenced the synthesis of Au NPs and thus demonstrated their ability to act as nanoreactors for the materials synthesis under different experimental conditions.
RESUMO
This study aimed to ensure the quality of the seed as well as determine the phytochemical composition of Nigella sativa seed extract (NSSE) obtained from three different geographical locations. Pharmacognostic evaluation of the seed includes preliminary phytochemical screening, physicochemical evaluation, and study of heavy metal content, in addition to HPTLC, HPLC, and GC-MS studies of the extract obtained from the seed of the Nigella sativa (NS). HPTLC fingerprinting studies revealed the presence of various bioactive compounds. HPLC analysis confirms the quantitative variation of thymoquinone (TQ) in the extracts, i.e. the maximum quantity of TQ was found in Vizag NSSE, followed by Punjab and Madhya Pradesh. GC-MS analysis reveals the presence of 33, 35, and 32 constituents in the extract obtained from Vizag, Madhya Pradesh, and Punjab, respectively. This study confirms the variation in the phytochemical composition as well as in the biomarker (Thymoquinone) content present in the collected samples.
RESUMO
The extent and magnitude of the mental health stigma are enormous, with substantial clinical and social implications. There is a complex relationship between mental health stigma and mental health professionals (MHPs); MHPs can be anti-stigma crusaders, victims of stigma, and even a source of stigma. Unfortunately, literature is scarce talking about the relationship between stigma and MHPs. Hence, the current review aims to bridge the existing gap in the literature on various aspects of stigma and the role of MHPs. For the current review, we ran a search in PubMed and Google Scholar databases; we restricted our study to records focusing on the interplay of mental health stigma and the MHPs, published during 2012-2022, in English, and having a full text available. We found that MHPs (psychiatrists, psychologists, and psychiatric nurses) can also be the recipients of the stigma. The stigma faced by the MHPs is determined by the negative stereotypes set by the media, or medical students, or other health professionals; the marginal position of psychiatry in the health system; difficult-to-treat mental disorders; MHPs' own experience of stigma; and the attitude or beliefs of various caders of the MHPs, their professional experience, and expertise in managing various mental health conditions. Notably, MHPs can also be a source of stigma (stigmatizers). MHPs need to be sensitized concerning this, and the anti-stigma interventions must incorporate this aspect of stigma. Novel interventions, such as digital-based programs, should be used instead of traditional anti-stigma programs in order to decrease stigma around mental health issues and make anti-stigma initiatives more appealing and scalable. To address the issues of stigma, there has to be more communication between MHPs, other health professionals, service users, and policymakers.
RESUMO
Memory deficits are observed across psychiatric disorders ranging from the prodrome of psychosis to common mental disorders such as anxiety, depression, and dissociative disorders. Memory deficits among patients recovering from psychiatric disorders could be directly related to the primary illness or secondary to the adverse effect of a treatment such as Electroconvulsive Therapy (ECT). The trouble in the meaningful integration of working-memory and episodic memory is the most commonly affected domain that requires routine assessments. An update on the recent trends of methods of assessment of memory deficits is the first step towards understanding and correcting these deficits to target optimum recovery. A systematic literature search was conducted from October 2018 to October 2022 to review the recent methods of assessment of memory deficits in psychiatric disorders. The definition of 'Memory deficit' was operationalized as 'selective processes of memory, commonly required for activities of daily living, and affected among psychiatric disorders resulting in subjective distress and dysfunction'. We included 110 studies, most of them being conducted in western countries on patients with schizophrenia. Other disorders included dementia and mild cognitive impairment. Brief Assessment of Cognition in Schizophrenia, Cambridge Automated Neuropsychological Test Battery, California Verbal Learning Test, Trail Making Test Part A and B, Rey Auditory Verbal Learning Test, Wechsler Memory Scale, Wechsler Adults Intelligence Scale-IV were the most common neuropsychological assessments used. Mini-Mental State Examination and Montreal Cognitive Assessment were the most common bedside assessment tools used while Squire Subjective Memory Questionnaire was commonly used to measure ECT-related memory deficits. The review highlights the recent developments in the field of assessment of memory deficits in psychiatric disorders. Findings recommend and emphasize routine assessment of memory deficits among psychiatric disorders in developing countries especially severe mental illnesses. It remains interesting to see the role of standardized assessments in diagnostic systems given more than a decade of research on memory deficits in psychiatric disorders.