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Two types of resonance-derived interfacial instability are reviewed with a focus on recent work detailing the effect of side walls on interfacial mode discretization. The first type of resonance is the mechanical Faraday instability, and the second is electrostatic Faraday instability. Both types of resonance are discussed for the case of single-frequency forcing. In the case of mechanical Faraday instability, inviscid theory can forecast the modal forms that one might expect when viscosity is taken into account. Experiments show very favourable validation with theory for both modal forms and onset conditions. Lowering of gravity is predicted to shift smaller wavelengths or choppier modes to lower frequencies. This is also validated by experiments. Electrostatic resonant instability is shown to lead to a pillaring mode that occurs at low wavenumbers, which is akin to Rayleigh Taylor instability. As in the case of mechanical resonance, experiments show favourable validation with theoretical predictions of patterns. A stark difference between the two forms of resonance is the observation of a gradual rise in the negative detuning instability in the case of mechanical Faraday and a very sharp one in the case of electrostatic resonance. This article is part of the theme issue 'New trends in pattern formation and nonlinear dynamics of extended systems'.
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Background The Covid-19 pandemic and subsequent lockdown in India caused disruptions in cancer treatment due to the restriction on movement of patients. We aimed to maintain continuity in cancer treatment during the lockdown through teleconsultations. We tried to reach out to our patients using telephonic consultations by establishing a Teleconsult Centre facility run by a team of doctors and patient navigators. Methods We telephonically contacted all patients who had outpatient appointments from 23 March to 30 April 2020 at our centre through the Teleconsult Centre to understand their current circumstances, feasibility of follow-up, local resources and offered best possible alternatives to continue cancer treatment, if required. Results Of the 2686 patients scheduled for follow-up during this period, we could contact 1783 patients in 9 working days. Through teleconsultations, we could defer follow-ups of 1034 patients (57.99%, 95% confidence interval [CI] 55.6%-60.3%), thus reducing the need for patients to travel to the hospital. Change in systemic therapy was made in 75 patients (4.2%, 95% CI 3.3%-5.2%) as per the requirements and available resources. Symptoms suggestive of disease progression were picked up in 12 patients (0.67%, 95% CI 0.35%-1.17%), who were advised to meet local physicians. Conclusion Our study suggests that the majority of patients on follow-up can be managed with teleconsultation in times of crisis. Teleconsultation has the potential of being one of the standard methods of patient follow-up even during periods of normalcy.
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COVID-19 , Neoplasias , Telemedicina , Humanos , COVID-19/epidemiologia , COVID-19/prevenção & controle , Centros de Atenção Terciária , Pandemias , Controle de Doenças Transmissíveis , Índia/epidemiologia , Continuidade da Assistência ao Paciente , Neoplasias/epidemiologia , Neoplasias/terapiaRESUMO
Biological removal of nitrogen and phosphorous from wastewater conventionally involves multiple processing steps to satisfy the differing oxygen requirements of the microbial species involved. In this work, simultaneous nitrification, denitrification, and phosphorous removal from synthetic wastewater were achieved by the fungus Neurospora discreta in a single-step, biofilm-based, aerobic process. The concentrations of carbon, nitrogen, and phosphorous in the synthetic wastewater were systematically varied to investigate their effects on nutrient removal rates and biofilm properties. Biofilm growth was significantly (p < 0.05) affected by carbon and nitrogen, but not by phosphorous concentration. Scanning electron microscopy revealed the effects of nutrients on biofilm microstructure, which in turn correlated with nutrient removal efficiencies. The carbohydrate and protein content in the biofilm matrix decreased with increasing carbon and nitrogen concentrations but increased with increasing phosphorous concentration in the wastewater. High removal efficiencies of carbon (96%), ammonium (86%), nitrate (100%), and phosphorus (82%) were achieved under varying nutrient conditions. Interestingly, decreasing the phosphorus concentration increased the nitrification and denitrification rates, and decreasing the nitrogen concentration increased the phosphorus removal rates significantly (p < 0.05). Correlations between biofilm properties and nutrient removal rates were also evaluated in this study.
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Nitrificação , Águas Residuárias , Desnitrificação , Eliminação de Resíduos Líquidos , Reatores Biológicos/microbiologia , Fósforo/metabolismo , Nitrogênio , Biofilmes , CarbonoRESUMO
Jaggery is a kind of unrefined non-centrifugal sugar (NCS) used mainly in Asia, Africa, Latin America, and the Caribbean. Traditionally, jaggery is produced by concentrating sugarcane juice in open pans with the help of bagasse combustion. However, due to thermal energy loss with flue gases and an unscientific approach in plant construction, jaggery plants have a poor thermal efficiency of less than 25%, poor emission characteristics, and a high bagasse consumption rate. Advanced jaggery-making techniques use solar energy and heat pumps for jaggery production. However, these techniques are in the early stage of development, and the literature indicates that these techniques should be used in conjuction with traditional ones to improve the performance of jaggery making plants. This literature review describes advances in jaggery-making methods, critically analyzed them, and provides a qualitative comparison of these methods. Further, gaps in the existing literature are identified and reported for future research direction. In addition, efforts have been made to quantify and estimate the emissions reduction and bagasse consumption potentials from the traditional jaggery industry to make this rural industry a sustainable and profitable business for rural entrepreneurs. The comparison with the recently developed clean combustion device exhibits that the harmful emissions from the jaggery industry could be reduced drastically viz. 95%-98% of PM2.5; 92%-95% of CO, and 52-60% of CO2, while saving more than 35% of bagasse consumption. Implemented at a national scale, it may reduce nearly 3% of all harmful emissions in the country, which is equally applicable elsewhere.
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Extratos Vegetais , Saccharum , Gases , Temperatura AltaRESUMO
Betel vine is an edible creeper used in folk medicine to aid digestion since time immemorial. It is an ideal candidate deemed for the bioprospection of endophytic microorganisms with valuable attributes. This study aimed at the characterization of potential bacteria from fermented betel leaves. We report the presence of Pediococcus species with probiotic properties from betel. The isolated organisms were subjected to preliminary biochemical analysis and exhibited growth at 37°C and pH 6.7 with fermented glucose, sucrose and lactose without the evolution of CO2. Also, the organisms presented tolerance to 6.5% NaCl and 0.3% bile salt. The three isolates assimilated cholesterol dispensed in the medium and when exposed to E. coli evinced antagonism. Based on the 16S rRNA sequencing and phylogenetic tree analysis, the organisms were identified to be Pediococcus acidilactici and Pediococcus pentosaceus. Both the organisms when functionally characterized displayed beta-galactosidase, amylase and esterase activities, but Pediococcus pentosaceus had a substantial effect proving its candidature for probiotic applications.
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Pediococcus , Piper betle , Escherichia coli , Pediococcus/genética , Filogenia , Folhas de Planta , RNA Ribossômico 16S/genéticaRESUMO
AIMS: To evaluate carbon source complexity as a process lever to impact the microstructure, chemical composition and water retention capacity of biofilms produced by Neurospora discreta. METHODS AND RESULTS: Biofilms were produced by nonpathogenic fungus N. discreta, using sucrose, cellulose or lignin as carbon source. The increase in complexity of carbon source from sucrose to lignin resulted in decreased water retention values (WRV) and wet weights of harvested biofilms. Confocal laser scanning microscopy was used to calculate porosity from bright-field images, and relative stained areas of cells and carbohydrates from fluorescence imaging of samples stained with Trypan blue and Alexa Fluor 488. Porosity and relative quantity of cells increased with increase in carbon source complexity while the amount of carbohydrates decreased. The chemical analysis of the extracted extracellular matrix (ECM) showed that biofilms grown on more complex carbon sources had lower carbohydrate and protein content, which also explains the lower WRV trend, as carbohydrates are hydrophilic. CONCLUSIONS: The nature of carbon source impacts the metabolic pathway of cells, thereby influencing the relative proportions of ECM and cells. This in turn impacts the microstructure, composition and water content of biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that carbon source can be used as process lever to control the properties of biofilms and presents a novel view of biofilms as potentially useful biomaterials.
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Biofilmes , Carbono/metabolismo , Matriz Extracelular/química , Neurospora/fisiologia , Carboidratos/química , Carbono/análise , Interações Hidrofóbicas e Hidrofílicas , Microscopia Confocal , Neurospora/química , Neurospora/metabolismo , Neurospora/ultraestrutura , Polissacarídeos/análise , Polissacarídeos/metabolismo , Porosidade , Água/análiseRESUMO
Biofilms are self-assembling structures consisting of rigid microbial cells embedded in a soft biopolymeric extracellular matrix (ECM), and have been commonly viewed as being detrimental to health and equipment. In this work, we show that biofilms formed by a non-pathogenic fungus Neurospora discreta, are fungal bio-composites (FBCs) that can be directed to self-organize through active stresses to achieve specific properties. We induced active stresses by systematically varying the agitation rate during the growth of FBCs. By growing FBCs that are strong enough to be conventionally tensile loaded, we find that as agitation rate increases, the elongation strain at which the FBCs break, increases linearly, and their elastic modulus correspondingly decreases. Using results from microstructural imaging and thermogravimetry, we rationalize that agitation increases the production of ECM, which concomitantly increases the water content of agitated FBCs up to 250% more than un-agitated FBCs. Water held in the nanopores of the ECM acts a plasticizer and controls the ductility of FBCs in close analogy with polyelectrolyte complexes. This paradigm shift in viewing biofilms as bio-composites opens up the possibility for their use as sustainable, biodegradable, low-modulus structural materials.
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Biofilmes , Módulo de Elasticidade , Interações Hidrofóbicas e Hidrofílicas , Neurospora/fisiologia , Biopolímeros/química , Matriz Extracelular/química , Matriz Extracelular/ultraestrutura , Nanoporos , Neurospora/química , Neurospora/ultraestrutura , Polieletrólitos/química , Resistência à TraçãoRESUMO
BACKGROUND: Diabetes, obesity and metabolic syndrome are highly prevalent in patients with severe mental illness and can impose a major physical health burden. OBJECTIVE: To determine how anthropometric and metabolic features changed over time in a retrospective cohort of people with Severe Mental Illness living in Cheshire, UK. METHODS: In all, 1307 individuals on the severe mental illness Register were followed up between 2002 and 2012 in UK general practice. Subjects were identified through a pseudanonymised search of general practice registers. RESULTS: Baseline body mass index was 28.6 kg/m2 increasing to 31.0 at 10-year follow-up ( r2 = 0.84; p = 0.0002). There was a significant increase in fasting blood glucose from 5.72 to 6.79 mmol/L ( r2 = 0.48; p = 0.026). Correspondingly, there was a strong positive univariate relation between increase in body mass index and fasting blood glucose ( r2 = 0.54; p < 0.0001) taking into account all measurements. Fasting blood glucose also increased slightly with age ( p = 0.028). With increasing use of statins, total cholesterol fell from 4.5 to 3.9 mmol/L ( r2 = 0.88; p = 0.0001), as did low-density lipoprotein cholesterol from 3.43 to 2.35 mmol/L ( r2 = 0.94; p = 0.0001). In multivariate models, adjusting for age, gender, smoking and blood pressure, each unit increase in body mass index (odds ratio = 1.07 [1.01, 1.13]; p = 0.031) and triglycerides (odds ratio = 1.28 (1.06, 1.55); p = 0.009) was independently associated with an increased risk of having type 2 diabetes. CONCLUSION: Increasing body mass index relates to increasing rates of dysglycaemia over time. Measures to encourage weight reduction should be key strategies to reduce dysglycaemia rates in severe mental illness. Prescribing statins may have been effective in improving the lipid profile in this group.
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Glicemia/metabolismo , Índice de Massa Corporal , Transtornos Mentais/metabolismo , Sistema de Registros , Idoso , Feminino , Seguimentos , Humanos , Masculino , Transtornos Mentais/sangue , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
It is shown that charged defect generation, through argon ion-based plasma processing, in few layer graphene, could substantially enhance the electrical capacitance for electrochemical energy storage. Detailed consideration of the constituent space charge and quantum capacitances were used to delineate a new length scale, correlated to electrically active defects contributing to the capacitance, and was found to be smaller than a structural correlation length determined through Raman spectroscopy. The study offers insights into an industrially viable method (i.e., plasma processing) for modifying and enhancing the energy density of graphene-based electrochemical capacitors.
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The density of states ϱ(E) of graphene is investigated numerically and within the self-consistent T-matrix approximation in the presence of vacancies within the tight binding model. The focus is on compensated disorder, where the concentration of vacancies n(A) and n(B) in both sublattices is the same. Formally, this model belongs to the chiral symmetry class BDI. The onlinear sigma model predicts for BDI a Gade-type singularity ϱ(E)â¼|E|(-1)exp[-|log(E)|(-1/x)]. Our numerical data are comparable to this result in a preasymptotic regime that gives way, however, at even lower energies to ϱ(E)â¼E(-1)|log(E)|(-xÌ), 1≤xÌ<2. We take this finding as evidence that, similar to the case of dirty d-wave superconductors, generic bipartite random hopping models may also exhibit unconventional (strong-coupling) fixed points for certain kinds of randomly placed scatterers if these are strong enough. Our research suggests that graphene with (effective) vacancy disorder is a physical representative of such systems.
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Fenneropenaeus indicus translationally controlled tumor protein (Fi-TCTP) was cloned and expressed using pET 100a-D-TOPO in prokaryotic expression system and it exhibited putative antioxidant activity as assessed in vitro by enhanced growth of Escherichia coli (E. coli) in presence of hydrogen peroxide. The protective efficacy of recombinant Fi-TCTP (rFi-TCTP) was evaluated in F. indicus by intramuscular and oral administration. Intramuscular injection of rFi-TCTP to shrimps, on subsequent white spot syndrome virus (WSSV) infection exhibited 42% relative percent survival. To understand the mechanism of protection, immunological parameters such as reactive oxygen species (ROS), phenoloxidase and mitochondrial membrane potential (MMP) were assessed in early (24h) and late (60h) stages of infection. rFi-TCTP pretreatment significantly lowers the WSSV induced ROS generation and respiratory burst during early and late stages of infection. Further, WSSV induced apoptotic changes such as reduced haemocyte count, loss in MMP and DNA fragmentation were significantly reduced during early and late stage of infection upon rFi-TCTP administration. Hence, the immunomodulatory studies suggest that protective effect of rFi-TCTP in treated shrimps, might be due to the reduction in ROS and apoptosis, following decreased mitochondrial damage together with reduced phenoloxidase activity and respiratory burst.
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Biomarcadores Tumorais/imunologia , Penaeidae/imunologia , Penaeidae/virologia , Animais , Apoptose/imunologia , Fragmentação do DNA , Oxirredução , Estresse Oxidativo/imunologia , Proteínas Recombinantes/imunologia , Proteína Tumoral 1 Controlada por Tradução , Vírus da Síndrome da Mancha Branca 1RESUMO
The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable 'enzyme-product' complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (Kb) values between 10.23 × 10(3) and 6.52 × 10(3) M(-1). The binding reactions were enthalpy driven with ΔHb values between -50.99 and -46.07 kJ mol(-1). From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.
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Antígenos de Helmintos/metabolismo , Ácido Aspártico Proteases/antagonistas & inibidores , Brugia Malayi/química , Proteínas de Helminto/metabolismo , Inibidores de Proteases/farmacologia , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/isolamento & purificação , Ácido Aspártico Proteases/metabolismo , Físico-Química , Relação Dose-Resposta a Droga , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificação , Humanos , Cinética , Estrutura Molecular , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , TemperaturaRESUMO
We report ATP1A3-associated rapid-onset dystonia-parkinsonism with an atypical presentation including myoclonus and exaggerated startle in four patients. Their prominence over parkinsonism prompted consideration of a syndromic diagnosis of myoclonus dystonia. ATP1α3 dysfunction in GABAergic neurons could explain these examination findings. The spectrum of ATP1A3-associated movement disorders includes myoclonus-dystonia.
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Distonia , Distúrbios Distônicos , Mioclonia , Transtornos Parkinsonianos , Humanos , Distonia/complicações , Mioclonia/complicações , Mioclonia/diagnóstico , Mutação , Distúrbios Distônicos/complicações , Distúrbios Distônicos/diagnóstico , Distúrbios Distônicos/genética , Transtornos Parkinsonianos/complicações , Transtornos Parkinsonianos/genética , ATPase Trocadora de Sódio-PotássioRESUMO
A series of 3-[3-(substituted phenyl)-1-phenyl-1H-pyrazol-5-yl]-2H-chromen-2-one (4a-k) were synthesized by reaction of 3-[2,3-dibromo-3-(substituted phenyl)propanoyl]-2H-chromen-2-one (3 a-k) with phenyl hydrazine in presence of triethylamine in absolute ethanol, characterized by spectral data and screened for their in vitro antibacterial activity against gram-positive and gram-negative bacteria. Among the series, compounds 4d, 4h and 4i displayed an encouraging antibacterial activity profile as compared to reference standard drug ciprofloxacin against tested bacterial strains.
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Antibacterianos/síntese química , Bacillus subtilis/efeitos dos fármacos , Benzopiranos/síntese química , Escherichia coli/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pirazóis/síntese química , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/farmacologia , Bacillus subtilis/crescimento & desenvolvimento , Benzopiranos/farmacologia , Ciprofloxacina/farmacologia , Escherichia coli/crescimento & desenvolvimento , Etanol/química , Etilaminas/química , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Fenil-Hidrazinas/química , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pirazóis/farmacologia , Staphylococcus aureus/crescimento & desenvolvimento , Relação Estrutura-AtividadeRESUMO
Innate immunity is the first line of defense in shrimps against invading pathogenic microorganisms. Peroxiredoxins (PRX) are the family of antioxidant proteins that play a crucial role in reduction of oxidative stress in host during viral infection. Peroxiredoxin from Fenneropenaeus indicus was identified, cloned and expressed in prokaryotic expression system. The Fi-PRX protein consists of two conserved Cys residues and belongs to typical 2-cys PRX family. The phylogenetic analysis revealed a close evolutionary relatedness of Fi-PRX with the PRX from Drosophila melanogaster PRX1 and distant origin with PRX sequences from other shrimp isolates Fenneropenaeus chinensis, Litopenaeus vannamei and Penaeus japonicus. Fi-PRX transcripts are constitutively expressed in hemocytes and tissues (gills, heart and muscle) and down regulated during 12 h, 24 h and 48 h of WSSV challenged shrimps. Fi-PRX protein levels correlated well with the corresponding levels of Fi-PRX transcripts in hemocytes and tissues of WSSV challenged shrimps. Recombinant Fi-PRX reduces insulin only in the presence of DTT suggesting that the antioxidant function of the protein is thiol dependent. These findings suggest that antioxidant activity of Fi-PRX play a significant role in neutralization of excessive free radicals and ROS generated during viral invasion.
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Antioxidantes/metabolismo , Infecções por Vírus de DNA/enzimologia , Penaeidae/virologia , Peroxirredoxinas/metabolismo , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Sequência de Aminoácidos , Animais , Antioxidantes/química , Sequência de Bases , Clonagem Molecular , Infecções por Vírus de DNA/genética , Infecções por Vírus de DNA/virologia , Ditiotreitol/metabolismo , Evolução Molecular , Expressão Gênica , Brânquias/enzimologia , Brânquias/virologia , Coração/virologia , Hemócitos/enzimologia , Hemócitos/virologia , Insulina/metabolismo , Dados de Sequência Molecular , Músculo Esquelético/enzimologia , Músculo Esquelético/virologia , Miocárdio/enzimologia , Oxirredução , Penaeidae/enzimologia , Penaeidae/imunologia , Peroxirredoxinas/química , Peroxirredoxinas/genética , Filogenia , Proteínas Recombinantes/farmacologia , Vírus da Síndrome da Mancha Branca 1/imunologiaRESUMO
Human lymphatic filariasis is a debilitating parasitic disease characterized by downregulation of the host's immune response in asymptomatic carriers along with profound hyperreactivity in chronic patients apart from putatively immune endemic normals. The endosymbiont Wolbachia, a bacterium of filarial nematodes has received much attention as possible chemotherapeutic target and its involvement in disease pathogenesis. The role of recombinant Wolbachia surface protein (rWSP), one of the most abundantly expressed proteins of the endosymbiont, in modulating cell-mediated immune responses in patients harboring Wuchereria bancrofti infections was evaluated in the current study. rWSP-induced lymphoproliferation with peripheral blood mononuclear cells suggested an impaired proliferative response in asymptomatic microfilaremic (MF) and symptomatic chronic pathology (CP) patients compared to endemic normals (EN). This was further supported by a significantly diminished expression of CD69 along with elevated levels of CD127 and CD62L in filarial patients (MF and CP) compared to EN. Further, rWSP induced the expression of regulatory T cell markers CTLA-4 and CD25 along with suppressor cytokines IL-10 and TGF-ß in MF and CP patients compared to EN. However, the rWSP-stimulated expression of IFN-γ was diminished significantly in filarial patients compared to endemic normals. Thus, these findings suggest that WSP may also contribute to the suppression of immune responses seen in filarial patients.
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Proteínas da Membrana Bacteriana Externa/imunologia , Filariose Linfática/imunologia , Proteínas de Membrana/imunologia , Linfócitos T/imunologia , Wolbachia/imunologia , Wuchereria bancrofti/microbiologia , Animais , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Antígeno CTLA-4/análise , Proliferação de Células , Citocinas/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/análise , Subunidade alfa de Receptor de Interleucina-7/análise , Selectina L/análise , Lectinas Tipo C/análise , Leucócitos Mononucleares/química , Leucócitos Mononucleares/imunologia , Linfócitos T/química , Linfócitos T Reguladores/química , Linfócitos T Reguladores/imunologia , Wuchereria bancrofti/patogenicidadeRESUMO
Very-early-onset IBD and infantile-onset IBD is extremely rare in children. There is paucity of data with regards to clinical profile and outcome of children with infantile-onset IBD from India. The clinicolaboratory profile, molecular genetic testing and treatment details of 8 children diagnosed with monogenic infantile-onset IBD during 2015-2020 is described here. The median age at onset of symptoms was 3 mo. Sibling death and consanguinity were noted in 4 (50%) each respectively. Diarrhea was the presentation in all (100%) and hematochezia in 5 (62%). Colonic ulcers on colonosopy was seen in 7 infants. The common mutation identified was IL-10R gene in 3 (42%) and LRBA gene mutation in 2 (25%). HSCT was done in 4 children and the rest were managed conservatively. Although there was no mortality in this series, two children (25%) were lost for follow-up.
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Colite Ulcerativa , Doenças Inflamatórias Intestinais , Proteínas Adaptadoras de Transdução de Sinal , Idade de Início , Criança , Colite Ulcerativa/diagnóstico , Consanguinidade , Humanos , Lactente , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/terapia , MutaçãoRESUMO
Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33 kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.
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Brugia Malayi/enzimologia , Clonagem Molecular/métodos , Corpos de Inclusão/química , Pepsina A/antagonistas & inibidores , Inibidores de Proteases/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Western Blotting , Brugia Malayi/genética , Caseínas/metabolismo , Cromatografia em Gel , Dicroísmo Circular , Filariose Linfática/metabolismo , Filariose Linfática/parasitologia , Escherichia coli , Glutaral/química , Humanos , Corpos de Inclusão/metabolismo , Cinética , Pepsina A/metabolismo , Plasmídeos , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Redobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Transformação BacterianaRESUMO
Carbophosphazene-based coordination ligands [{NC(NMe(2))}(2){NP(3,5-Me(2)Pz)(2)}] (1), [{NC(NEt)(2)}{NC(3,5-Me(2)Pz)}{NP(3,5-Me(2)Pz)(2)}] (2), [NC(3,5-Me(2)Pz)](2)[NP(3,5-Me(2)Pz)(2)] (3), [{NCCl}(2){NP(NC(NMe(2))(2))(2)}] (4), and [{NC(p-OC(5)H(4)N)}(2){NP(NC(NMe(2))(2))(2)}] (5) were synthesized and structurally characterized. In these compounds, the six-membered C(2)N(3)P ring is perfectly planar. The reaction of 1 with CuCl(2) afforded [{NC(NMe(2))}(2){NHP(O)(3,5-Me(2)Pz)}·{Cu(3,5-Me(2)PzH)(2)(Cl)}][Cl] (6). The ligand binds to Cu(II) utilizing the geminal [P(O)(3,5-Me(2)Pz)] coordinating unit. Similarly, the reaction of 2 with PdCl(2) afforded, after a metal-assisted P-N hydrolysis, [{NC(NEt)(2)}{NC(3,5-Me(2)Pz)}{NP(O)(3,5-Me(2)Pz)}·{Pd(3,5-Me(2)PzH)(Cl)}] (7). In the latter, the [P(O)(3,5-Me(2)Pz)] unit does not coordinate; in this instance, the Pd(II) is bound by a ring nitrogen atom and a carbon-tethered pyrazolyl nitrogen atom. The reaction of 3 with PdCl(2) also results in P-N bond hydrolysis affording [{NC(3,5-Me(2)Pz)(2)}{NP(O)(3,5-Me(2)Pz)}{Pd(Cl)}] (8). In contrast to 7, however, in 8, the Pd(II) elicits a nongeminal η(3) coordination from the ligand involving two carbon-tethered pyrazolyl groups and a ring nitrogen atom. Metalated products could not be isolated in the reaction of 3 with K(2)PtCl(4). Instead, a P-O-P bridged carbodiphosphazane dimer, [{NC(3,5-Me(2)Pz)NHC(3,5-Me(2)Pz)}{NP(O)}](2) (9), was isolated as the major product. Finally, the reaction of 5 with PdCl(2) resulted in [{NC(OC(5)H(4)N)}(2){NP(NC(NMe(2))(2))(2)}·{PdCl(2)}] (10). In the latter, the exocyclic P-N bonds are quite robust and are involved in binding to the metal ion. Compounds 6-10 have been characterized by a variety of techniques including X-ray crystallography. In all of the compounds, the bond parameters of the inorganic heterocyclic rings are affected by metalation.
Assuntos
Guanidina/química , Compostos Organofosforados/química , Pirazóis/química , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Estrutura Molecular , Compostos Organofosforados/síntese química , EstereoisomerismoRESUMO
Immune responses to recombinant Brugia malayi pepsin inhibitor homolog (rBm-33) were investigated in patients with human lymphatic filariasis (microfilaremics (MF) and chronic pathology (CP)) along with endemic normals (EN). Flow cytometric analysis (24 h) revealed CD4(+) T cell activation in patients (MF and CP) compared to normals (EN), with increased expression of CD69 and diminished levels of CD62L and CD127. This was associated with an elevated expression of CD154 but not CD28 and CTLA4 in CP patients. However, Bm-33-induced cytokine expression profile (IL-1ß, IL-12, IL-8, IFN-γ, IL-10 and TGF-ß) did not exhibit any significant difference between normals and patients at the same time point. Although CD4(+) T cell activation was observed initially in filarial patients (24 h), lymphoproliferation studies (96 h) suggested diminished proliferation compared to normals, indicating functional inactivation in the former upon prolonged antigen exposure. This indicates that rBm-33 induces an early T cell activation in MF and CP patients followed by a decreased lymphoproliferation that might contribute to immune suppression in these individuals.