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AIM: This study is aimed to evaluate the combined effect of sodium hypochlorite at varied concentrations and temperatures on radicular dentin microhardness along with its surface structural changes using an FTIR spectrometer. MATERIALS AND METHODS: Mandibular premolars were cleaned and shaped up to F3 Protaper gold rotary files, after which they were subjected to five experimental conditions - group I - neutral saline as negative control, group II - 3% NaOCl solution, group III - 5% NaOCl solution, group IV - 3% intracanal-heated NaOCl solution, and group V - 5% intracanal-heated NaOCl solution. Following this, the microhardness of radicular dentin at 100 µm and 300 µm from the canal lumen and Fourier-transform infrared (FTIR) spectroscopic analysis were performed. RESULTS: The results showed that intracanal-heated sodium hypochlorite group reduced root dentin microhardness at 300 µm than its nonheated counterpart. No difference in microhardness values was observed between 3% intracanal-heated and room-temperature sodium hypochlorite groups at 100 µm. Reduction in amide/phosphate ratio was noted in all the groups treated with sodium hypochlorite irrespective of temperature and concentration. CONCLUSION: Thus, considering that the level of alteration in physical and structural changes of root dentin with or without heating is insignificant, intracanal-heated low-concentration sodium hypochlorite solutions could be used as an alternative to high-concentration sodium hypochlorite. CLINICAL SIGNIFICANCE: Intracanal-heated low-concentration sodium hypochlorite enables the clinicians to achieve maximum disinfection while keeping the structural and physical properties of the dentin similar to room-temperature sodium hypochlorite.
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Cavidade Pulpar , Hipoclorito de Sódio , Hipoclorito de Sódio/farmacologia , Sódio/farmacologia , Irrigantes do Canal Radicular/farmacologia , Dentina , Ácido EdéticoRESUMO
Repeated exposure to adverse experiences in early life, termed Early Life Stress (ELS), can increase anxiety disorders later in life. Anxiety is directly associated with curiosity, a form of intrinsic drive state associated with increased novelty-seeking behaviour and risk taking for challenging opportunities and could probably modulate learning and memory. In humans, elevated curiosity during adolescence tends to elicit increased exploration, novelty seeking, high risk-taking behaviour and heightened emotionality. Such behaviours are beneficial in maintaining social skills and cognitive functions later in life. We investigated whether ELS-induced anxiety impacts curiosity-like behaviour at adolescence in an animal model. ELS was induced by subjecting Sprague Dawley rat pups to maternal separation and isolation (MS) stress during the stress hyporesponsive period (SHRP) from post-natal days (PND) 4-PND 14. This rat model was tested for anxiety, spontaneous exploratory behaviour and curiosity-like behaviour in a custom-designed arena during adolescence (PND 30-45). ELS-induced changes in the stress were confirmed by corticosterone, while, basal dopamine level was estimated to understand the neurochemical basis of MS stress-induced changes in curiosity. We observed an increase in the levels of anxiety and intrinsic drive state such as curiosity-like behaviour, which was associated with elevated plasma corticosterone and dopamine in MS animals during adolescence suggesting the impact of ELS during SHRP on adolescent behaviour.
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Experiências Adversas da Infância , Comportamento Exploratório , Animais , Ansiedade/etiologia , Ansiedade/psicologia , Transtornos de Ansiedade , Corticosterona , Dopamina , Humanos , Privação Materna , Ratos , Ratos Sprague-Dawley , Estresse Psicológico/psicologiaRESUMO
DNA cytosine modifications are key epigenetic regulators of cellular processes in mammalian cells, with their misregulation leading to varied disease states. In the human malaria parasite Plasmodium falciparum, a unicellular eukaryotic pathogen, little is known about the predominant cytosine modifications, cytosine methylation (5mC) and hydroxymethylation (5hmC). Here, we report the first identification of a hydroxymethylcytosine-like (5hmC-like) modification in P. falciparum asexual blood stages using a suite of biochemical methods. In contrast to mammalian cells, we report 5hmC-like levels in the P. falciparum genome of 0.2-0.4%, which are significantly higher than the methylated cytosine (mC) levels of 0.01-0.05%. Immunoprecipitation of hydroxymethylated DNA followed by next generation sequencing (hmeDIP-seq) revealed that 5hmC-like modifications are enriched in gene bodies with minimal dynamic changes during asexual development. Moreover, levels of the 5hmC-like base in gene bodies positively correlated to transcript levels, with more than 2000 genes stably marked with this modification throughout asexual development. Our work highlights the existence of a new predominant cytosine DNA modification pathway in P. falciparum and opens up exciting avenues for gene regulation research and the development of antimalarials.
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5-Metilcitosina/análogos & derivados , DNA de Protozoário/genética , Epigênese Genética , Genoma de Protozoário , Plasmodium falciparum/genética , RNA Mensageiro/genética , 5-Metilcitosina/metabolismo , Citosina/metabolismo , Metilação de DNA , DNA de Protozoário/metabolismo , Eritrócitos/parasitologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hidroxilação , Plasmodium falciparum/metabolismo , RNA Mensageiro/metabolismoRESUMO
The electrochemistry and photophysics of the Pt(II) complexes [Pt(naphen)(X)] (Hnaphen = naphtho[1,2-b][1,10]phenanthroline, X = Cl or C≡CPh) containing the rigid tridentate C^N^N-coordinating pericyclic naphen ligand was studied alongside the complexes of the tetrahydro-derivative [Pt(thnaphen)(X)] (Hthnaphen = 5,6,8,9-tetrahydro-naphtho[1,2-b][1,10]phenanthroline) and the N^C^N-coordinated complex [Pt(bdq)(Cl)] (Hbdq = benzo[1,2-h:5,4-h']diquinoline. The cyclic voltammetry showed reversible reductions for the C^N^N complexes, with markedly fewer negative potentials (around -1.6 V vs. ferrocene) for the complexes containing the naphen ligand compared with the thnaphen derivatives (around -1.9 V). With irreversible oxidations at around +0.3 V for all of the complexes, the naphen made a difference in the electrochemical gap of about 0.3 eV (1.9 vs. 2.2 eV) compared with thnaphen. The bdq complex was completely different, with an irreversible reduction at around -2 V caused by the N^C^N coordination pattern, which lacked a good electron acceptor such as the phenanthroline unit in the C^N^N ligand naphen. Long-wavelength UV-Vis absorption bands were found around 520 to 530 nm for the C^N^N complexes with the C≡CPh coligand and were red-shifted when compared with the Cl derivatives. The N^C^N-coordinated bdq complex was markedly blue-shifted (493 nm). The steady-state photoluminescence spectra showed poorly structured emission bands peaking at around 630 nm for the two naphen complexes and 570 nm for the thnaphen derivatives. The bdq complex showed a pronounced vibrational structure and an emission maximum at 586 nm. Assuming mixed 3LC/3MLCT excited states, the vibronic progression for the N^C^N bdq complex indicated a higher LC character than assumed for the C^N^N-coordinated naphen and thnaphen complexes. The blue-shift was a result of the different N^C^N vs. C^N^N coordination. The photoluminescence lifetimes and quantum yields ΦL massively increased from solutions at 298 K (0.06 to 0.24) to glassy frozen matrices at 77 K (0.80 to 0.95). The nanosecond time-resolved study on [Pt(naphen)(Cl)] showed a phosphorescence emission signal originating from the mixed 3LC/3MLCT with an emission lifetime of around 3 µs.
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Photoactive metal complexes containing earth-abundant transition metals recently gained interest as photosensitizers in light-driven chemistry. In contrast to the traditionally employed ruthenium or iridium complexes, iron complexes developed to be promising candidates despite the fact that using iron complexes as photosensitizers poses an inherent challenge associated with the low-lying metal-centered states, which are responsible for ultrafast deactivation of the charge-transfer states. Nonetheless, recent developments of strongly σ-donating carbene ligands yielded highly promising systems, in which destabilized metal-centered states resulted in prolonged lifetimes of charge-transfer excited states. In this context, we introduce a series of novel homoleptic Fe-triazolylidene mesoionic carbene complexes. The excited-state properties of the complexes were investigated by time-resolved femtosecond transient absorption spectroscopy and quantum chemical calculations. Pump wavelength-dependent transient absorption reveals the presence of distinct excited-state relaxation pathways. We relate the excitation-wavelength-dependent branching of the excited-state dynamics into various reaction channels to solvent-dependent photodissociation following the population of dissociative metal centered states upon excitation at 400 nm.
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BACKGROUND: Transmission of multidrug-resistant tuberculosis (MDRTB) requires spatial proximity between infectious cases and susceptible persons. We assess activity space overlap among MDRTB cases and community controls to identify potential areas of transmission. METHODS: We enrolled 35 MDRTB cases and 64 TB-free community controls in Lima, Peru. Cases were whole genome sequenced and strain clustering was used as a proxy for transmission. GPS data were gathered from participants over seven days. Kernel density estimation methods were used to construct activity spaces from GPS locations and the utilization distribution overlap index (UDOI) was used to quantify activity space overlap. RESULTS: Activity spaces of controls (median = 35.6 km2, IQR = 25.1-54) were larger than cases (median = 21.3 km2, IQR = 17.9-48.6) (P = 0.02). Activity space overlap was greatest among genetically clustered cases (mean UDOI = 0.63, sd = 0.67) and lowest between cases and controls (mean UDOI = 0.13, sd = 0.28). UDOI was positively associated with genetic similarity of MDRTB strains between case pairs (P < 0.001). The odds of two cases being genetically clustered increased by 22% per 0.10 increase in UDOI (OR = 1.22, CI = 1.09-1.36, P < 0.001). CONCLUSIONS: Activity space overlap is associated with MDRTB clustering. MDRTB transmission may be occurring in small, overlapping activity spaces in community settings. GPS studies may be useful in identifying new areas of MDRTB transmission.
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Infecções Comunitárias Adquiridas/epidemiologia , Infecções Comunitárias Adquiridas/transmissão , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/transmissão , Adulto , Feminino , Sistemas de Informação Geográfica , Humanos , Masculino , Pessoa de Meia-Idade , Peru/epidemiologia , Rede Social , Adulto JovemRESUMO
Cyclophosphamide (CP) is a potent chemotherapeutic agent and is also known to interact with specific biological molecules and produce numerous side effects such as genotoxicity, neurotoxicity, reproductive toxicity and nephrotoxicity. CP induces genotoxicity by generating oxygen/nitrogen derived free radicals during chemotherapy and causes DNA damage. Hence, to overcome such side effects of chemotherapeutic agents antioxidants are recommended. Gallic acid (GA) a phenolic compound is commonly exists in variety of fruits and in many plants. In the present study, genoprotecive effects of GA against CP induced genotoxicity in Swiss albino mice were evaluated by using comet assay, bone marrow, and peripheral micronucleus (MN) assays. CP produced oxidative stress induced hepatic damage was assessed by estimating the activity of liver superoxide dismutase (SOD), reduced glutathione content (GSH), and also by examining the histological alterations induced by CP in hepatic tissue of mice. It was noticed that, GA pretreatment significantly reduced the frequency of MN and DNA strand breaks induced by CP. GA also protected the hepatic tissue against CP induced reactive oxygen species (ROS) mediated damage and was evident by increased levels of liver SOD and GSH. GA ameliorated the histological changes induced by CP in hepatic tissue. These findings suggest that, GA is a versatile antioxidant with promising protection against CP induced genotoxicity and hepatic damage in Swiss albino mice.
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Protein folding in the endoplasmic reticulum (ER) is monitored by ER quality control (ERQC) mechanisms. Proteins that pass ERQC criteria traffic to their final destinations through the secretory pathway, whereas non-native and unassembled subunits of multimeric proteins are degraded by the ER-associated degradation (ERAD) pathway. During ERAD, molecular chaperones and associated factors recognize and target substrates for retrotranslocation to the cytoplasm, where they are degraded by the ubiquitin-proteasome machinery. The discovery of diseases that are associated with ERAD substrates highlights the importance of this pathway. Here, we summarize our current understanding of each step during ERAD, with emphasis on the factors that catalyse distinct activities.
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Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Ubiquitina/metabolismo , Animais , Citoplasma/metabolismo , Humanos , Dobramento de Proteína , Transporte Proteico , Via Secretória , Especificidade por Substrato , UbiquitinaçãoRESUMO
Background & objectives: Infection from fluoroquinolone-resistant extra-intestinal Escherichia coli is a global concern. In this study, isolation and characterization of fluoroquinolone-resistant extra-intestinal E. coli isolates obtained from hospital samples were undertaken to detect plasmid-mediated quinolone resistance (PMQR) genes. Methods: Forty three isolates of E. coli obtained from patients with extra-intestinal infections were subjected to antibiogram to detect fluoroquinolone resistance. The mechanism of fluoroquinolone resistance was determined by the detection of PMQR genes and mutations in quinolone resistance determining region (QRDR). Results: Of the 43 isolates, 36 were resistant to nalidixic acid (83.72%) and 28 to ciprofloxacin (65.11%). Eight E. coli isolates showed total resistance to both the antimicrobials without any minimum inhibitory concentration. The detection of PMQR genes with qnr primers showed the presence of qnrA in two, qnrB in six and qnrS in 21 isolates. The gene coding for quinolone efflux pump (qepA) was not detected in any of the isolates tested. The presence of some unexpressed PMQR genes in fluoroquinolone sensitive isolates was also observed. Interpretation & conclusions: The detection of silent PMQR genes as observed in the present study presents a risk of the transfer of the silent resistance genes to other microorganisms if present in conjugative plasmids, thus posing a therapeutic challenge to the physicians. Hence, frequent monitoring is to be done for all resistance determinants.
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Farmacorresistência Bacteriana/genética , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Fluoroquinolonas/efeitos adversos , Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Ciprofloxacina/efeitos adversos , Ciprofloxacina/farmacologia , DNA Topoisomerases/efeitos dos fármacos , DNA Topoisomerases/genética , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Fluoroquinolonas/uso terapêutico , Humanos , Plasmídeos/efeitos dos fármacos , Plasmídeos/genéticaRESUMO
Dormant liver stage forms (hypnozoites) of the malaria parasite Plasmodium vivax present major hurdles to control and eradicate infection. Despite major research efforts, the molecular composition of hypnozoites remains ill defined. Here, we applied a combination of state-of-the-art technologies to generate the first transcriptome of hypnozoites. We developed a robust laser dissection microscopy protocol to isolate individual Plasmodium cynomolgi hypnozoites and schizonts from infected monkey hepatocytes and optimized RNA-seq analysis to obtain the first transcriptomes of these stages. Comparative transcriptomic analysis identified 120 transcripts as being differentially expressed in the hypnozoite stage relative to the dividing liver schizont, with 69 and 51 mRNAs being up- or down-regulated, respectively, in the hypnozoites. This lead to the identification of potential markers of commitment to and maintenance of the dormant state of the hypnozoite including three transcriptional regulators of the ApiAP2 family, one of which is unique to P. cynomolgi and P. vivax, and the global translational repressor, eIF2a kinase eIK2, all of which are upregulated in the hypnozoite. Together, this work not only provides a primary experimentally-derived list of molecular markers of hypnozoites but also identifies transcriptional and posttranscriptional regulation of gene expression as potentially being key to establishing and maintaining quiescence.
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Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Fígado/parasitologia , Plasmodium cynomolgi/fisiologia , Animais , Haplorrinos , Hepatócitos/parasitologia , Microdissecção e Captura a LaserRESUMO
AIM: The present study was conducted to analyze the clinical and histopathological cases of odontogenic tumors (OTs). MATERIALS AND METHODS: The present 10-year retrospective study comprised of 104 OTs. Parameters such as name, age, gender, clinical features, location, extension, etc were noted. H and E stained slides were carefully assessed by an oral pathologist and were classified according to the latest WHO classification of head and neck tumors. RESULTS: Out of 104 OTs, the most common was ameloblastoma constituting 45 cases, KCOT (28), odontoma (17), odontogenic myxoma (4), Calcifying epithelial odontogenic tumor (CEOT) (5), cementoblastoma (3) and calcifying cystic odontogenic tumor (2). The p value found to be 0.01 (significant). Common OTs was ameloblastoma (25 males and 20 males), KCOT (12 males and 16 females), odontoma (10 males and 7 females), odontogenic myxoma (3 males and 1 female), CEOT (3 males and 2 females), cementoblastoma (2 males and 1 female) and calcifying cystic odontogenic tumor (1 male and 1 female). Ameloblastoma, KCOT, and odontoma were predominantly seen in the age group 21-30 years, CEOT and cementoblastoma in age group 31-40 years. The difference was significant (P < 0.05). Common clinical features in OTs were facial disfigurement (65), swelling (78) and pain (55). The difference was non significant (P > 0.05). The average size of ameloblastoma was 6.8cm, KCOT was 4.2 cm, odontoma was 3.9 cm, odontogenic myxoma was 2.7 cm, CEOT was 5.5 cm, cementoblastoma was 3.8 cm and Calcifying cystic odontogenic tumour (COC) was 3.6 cm. The difference was non-significant (p > 0.05). CONCLUSION: Mandible exhibited more OTs as compared to the maxilla. The most common lesion was ameloblastoma, KCOT, and odontomas. We observed male predominance. CLINICAL SIGNIFICANCE: The study helps in assessing the occurrence of the odontogenic tumor. This is useful for identification and clinical management.
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Neoplasias Mandibulares/patologia , Neoplasias Maxilares/patologia , Tumores Odontogênicos/patologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Índia/epidemiologia , Lactente , Recém-Nascido , Masculino , Neoplasias Mandibulares/epidemiologia , Neoplasias Maxilares/epidemiologia , Pessoa de Meia-Idade , Tumores Odontogênicos/epidemiologia , Estudos Retrospectivos , Fatores Sexuais , Fatores de Tempo , Adulto JovemRESUMO
Cytotoxic T lymphocytes (CTL) eliminate pathogen-infected and cancerous cells mainly by polarized secretion of lytic granules (LG, containing cytotoxic molecules like perforin and granzymes) at the immunological synapse (IS). Members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family are involved in trafficking (generation, transport and fusion) of vesicles at the IS. Syntaxin 8 (Stx8) is expressed in LG and colocalizes with the T cell receptor (TCR) upon IS formation. Here, we report the significance of Stx8 for human CTL cytotoxicity. We found that Stx8 mostly localized in late, recycling endosomal and lysosomal compartments with little expression in early endosomal compartments. Down-regulation of Stx8 by siRNA resulted in reduced cytotoxicity. We found that following perforin release of the pre-existing pool upon target cell contact, Stx8 down-regulated CTL regenerate perforin pools less efficiently and thus release less perforin compared to control CTL. CD107a degranulation, real-time and end-point population cytotoxicity assays, and high resolution microscopy support our conclusion that Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules to functional LG through the endosomal pathway in human CTL.
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Grânulos Citoplasmáticos/metabolismo , Proteínas Qa-SNARE/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/metabolismo , Degranulação Celular , Linhagem Celular , Grânulos Citoplasmáticos/imunologia , Citotoxicidade Imunológica , Endossomos/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Perforina/metabolismo , Transporte Proteico , Proteínas Qa-SNARE/genética , Interferência de RNA , Vesículas Secretórias/imunologia , Linfócitos T Citotóxicos/imunologia , Fatores de Tempo , TransfecçãoRESUMO
OBJECTIVE: To study the acute phase serum biomarkers in patients with mild traumatic brain injury (mTBI) and to correlate them with short term cognitive deficits. MATERIALS AND METHODS: This is a prospective observational study conducted at a tertiary care center for neurotrauma. The participants included patients with mTBI (n = 20) and age, gender, and education-status matched healthy controls (n = 20). In both the groups, the serum concentrations of biomarkers ubiquitin C terminal hydrolase (UCH-L1) and S100 calcium-binding protein B (S100B) were measured. Both the groups underwent neuropsychological tests. The serum tests were done in the acute stage after injury and the neuropsychological tests were done 3 months after injury. RESULTS: There was no significant increase in the serum S100B and UCH-L1 levels in patients with mTBI. Patients with mTBI had significant cognitive deficits at 3 months after injury, which was suggestive of involvement of diffuse areas of the brain, in particular, the premotor, prefrontal, and medial inferior frontal lobes and the basitemporal region. The correlation of biomarkers with cognitive deficits in patients with mTBI was found in the following domains: working memory, verbal learning, verbal fluency, and visual memory. CONCLUSION: The serum biomarkers of mTBI have a correlation with selective domains of neuropsychological outcome.
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Biomarcadores/sangue , Concussão Encefálica/sangue , Disfunção Cognitiva/sangue , Subunidade beta da Proteína Ligante de Cálcio S100/sangue , Ubiquitina Tiolesterase/sangue , Adolescente , Adulto , Concussão Encefálica/complicações , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/etiologia , Feminino , Humanos , Masculino , Síndrome Pós-Concussão/sangue , Estudos Prospectivos , Adulto JovemRESUMO
In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS.
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Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Lisossomos/metabolismo , Mitocôndrias/fisiologia , Extratos de Tecidos/farmacologia , Adulto , Esclerose Lateral Amiotrófica/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Injeções Espinhais , Masculino , Potencial da Membrana Mitocondrial , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas Mitocondriais/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/ultraestrutura , Estresse Oxidativo , Proteoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
We have studied in detail the situation wherein two microbeads are trapped axially in a single-beam Gaussian intensity profile optical trap. We find that the corner frequency extracted from a power spectral density analysis of intensity fluctuations recorded on a quadrant photodetector (QPD) is dependent on the detection scheme. Using forward- and backscattering detection schemes with single and two laser wavelengths along with computer simulations, we conclude that fluctuations detected in backscattering bear true position information of the bead encountered first in the beam propagation direction. Forward scattering, on the other hand, carries position information of both beads with substantial contribution from the bead encountered first along the beam propagation direction. Mie scattering analysis further reveals that the interference term from the scattering of the two beads contributes significantly to the signal, precluding the ability to resolve the positions of the individual beads in forward scattering. In QPD-based detection schemes, detection through backscattering, thereby, is imperative to track the true displacements of axially trapped microbeads for possible studies on light-mediated interbead interactions.
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In Plasmodium falciparum, perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Here we identify a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2, a TARE6-associated histone deacetylase linked to the process of antigenic variation. The nuclear location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation.
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Proteínas de Ligação a DNA/metabolismo , Plasmodium falciparum/genética , Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Arqueais/química , Citoplasma/química , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Dimerização , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/ultraestrutura , Estrutura Terciária de Proteína , Proteínas de Protozoários/análise , Proteínas de Protozoários/química , RNA/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/química , Sequências Repetitivas de Ácido Nucleico , Telômero/químicaRESUMO
A hybrid hydrogel nanocomposite derived from cellulose fiber extracted from Banana Pseudo Stem (BPS) was developed as an adsorbent material for wastewater treatment. The hydrogel was developed by graft copolymerization of N-hydroxyethylacrylamide on Cellulose Fiber (BPSCF-g-PHEAAm) with potassium peroxodisulphate (KPS) as an initiator and N, N'-methylene bisacrylamide (MBA) as a crosslinker using microwave irradiation. Magnetic nanoparticles generated by an in-situ method were incorporated into the network structure. Fourier Transform Infrared Spectroscopy (FTIR), Powder X-ray Diffraction (XRD), Thermogravimetric analysis (TGA), Vibrating Sample Magnetometer (VSM), Brunauer-Emmett-Teller analysis (BET), Field Emission Scanning Electron Microscopy (FESEM), and Energy Dispersive Spectrometer (EDS) were employed. The adsorption capacities of hydrogel and its nanocomposite were evaluated using Methylene Blue (MB) and Crystal Violet (CV) as model dyes. The parent gel exhibited the maximum absorption capacity of 235, and 219 mg g-1 towards MB and CV respectively which was enhanced to 320 and 303 mg g-1 for the nanocomposite. Adsorption data were best fitted with the pseudo-second-order kinetic model and the Freundlich isotherm model. Negative ΔG° and positive ΔH° indicated spontaneous and endothermic adsorption. Desorption was effective to an extent of 99 % in the HCl medium suggesting high reusability potential of the developed adsorbent material.
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Light-inducible regulation of cellular pathways and gene circuits in mammalian cells is a new frontier in mammalian genetic engineering. Optogenetic mammalian cell cultures, which are light-sensitive engineered cells, utilize light to regulate gene expression and protein activity. As a low-cost, tunable, and reversible input, light is highly adept at spatiotemporal and orthogonal regulation of cellular behavior. However, light is absorbed and scattered as it travels through media and cells, and the applicability of optogenetics in larger mammalian bioreactors has not been determined. In this work, we computationally explore the size limit to which optogenetics can be applied in cylindrical bioreactors at relevant height-to-diameter ratios. We model the propagation of light using the radiative transfer equation and consider changes in reactor volume, absorption coefficient, scattering coefficient, and scattering anisotropy. We observe sufficient light penetration for activation in simulated bioreactors with sizes of up to 80,000 L at maximal cell densities. We performed supporting experiments and found that significant attenuation occurs at the boundaries of the system, but the relative change in intensity distribution within the reactor was consistent with simulation results. We conclude that optogenetics can be applied to bioreactors at an industrial scale and may be a valuable tool for specific biomanufacturing applications.
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Reatores Biológicos , Optogenética , Animais , Optogenética/métodos , Técnicas de Cultura de Células , Mamíferos , Contagem de CélulasRESUMO
Inorganic photoacids and photobases comprising of photoactive transition metal complexes (TMCs) offer the ability to modulate proton transfer reactions through light irradiation, while utilizing the excellent optical properties of the latter. This provides a powerful tool for precise control over chemical reactions and processes, with implications for both fundamental science and practical applications. In this contribution, we present a novel molecular architecture amending an Fe-NHC complex with a pendant quinoline, as a prototypical photobase, as a representative earth-abundant TMC based inorganic photobase. We characterize the excited-state properties and proton-transfer dynamics using steady-state absorption and emission spectroscopy as well as pump wavelength dependent transient absorption spectroscopy in various protic solvents. The kinetics and thermodynamics of proton transfer in the quinoline moiety are influenced by both the presence of the metal center and the choice of the solvent. Furthermore, we see indications of intramolecular energy transfer from the quinoline to the MLCT state as a limiting factor for panchromatic photobasicity of the complex.
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The major function of cytotoxic T lymphocytes (CTLs) is to eliminate pathogen-infected and tumorigenic cells. This is mediated mainly through the exocytosis of lytic granules (LGs) containing cytotoxic components, such as perforin and granzymes at the immunological synapse (IS). The soluble NSF attachment receptor (SNARE) protein isoforms are well known to be required for vesicle exocytosis in neuronal synapses, but their potential function in CTLs is only partly understood. Here, we examined the expression of SNARE proteins before and after the activation of primary human CD8(+) T cells and determined their co-localization with LGs and CD3 after IS formation with target cells. We found that several key SNARE proteins in neuronal cells were not expressed in CTLs, such as syntaxin1B2 and SNAP-25. Vti1b, Stx8 and Stx16 had the highest degrees of co-localization with LGs while Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 and VAMP4 co-localized with CD3. Our data provide the first complete expression profile and localization of SNAREs in primary human CD8(+) T cells, laying the groundwork for further understanding their potential role in T-cell function.