RESUMO
Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane ß-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane ß-hairpin region. KEY POINTS: ⢠Cry46Ab mutants were constructed by targeting the putative transmembrane ß-hairpin region. ⢠Charged residues within the ß-hairpin control the flux of ions through channel pores. ⢠Channel-pore cation selectivity is correlated with insecticidal activity.
Assuntos
Bacillus thuringiensis , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Cátions , Endotoxinas , Proteínas Hemolisinas/genética , Mutagênese Sítio-DirigidaRESUMO
OBJECTIVES: The objective of the current work was to evaluate flow and turbulent kinetic energy in different transcatheter aortic valve implants using highly undersampled time-resolved multi-point 3-directional phase-contrast measurements (4D Flow MRI) in an in vitro setup. MATERIALS AND METHODS: A pulsatile flow setup was used with a compliant tubing mimicking a stiff left ventricular outflow tract and ascending aorta. Five different implants were measured using a highly undersampled multi-point 4D Flow MRI sequence. Velocities and turbulent kinetic energy values were analysed and compared. RESULTS: Strong variations of turbulent kinetic energy distributions between the valves were observed. Maximum turbulent kinetic energy values ranged from 100 to over 500 J/m3 while through-plane velocities were similar between all valves. CONCLUSION: Highly accelerated 4D Flow MRI for the measurement of velocities and turbulent kinetic energy values allowed for the assessment of hemodynamic parameters in five different implant models. The presented setup, measurement protocol and analysis methods provides an efficient approach to compare different valve implants and could aid future novel valve designs.
Assuntos
Valva Aórtica , Técnicas de Imagem Cardíaca/métodos , Próteses Valvulares Cardíacas , Imageamento por Ressonância Magnética/métodos , Substituição da Valva Aórtica Transcateter , Valva Aórtica/cirurgia , Velocidade do Fluxo Sanguíneo , Hemorreologia , Humanos , Imageamento Tridimensional/métodos , Técnicas In Vitro , Imagem Cinética por Ressonância Magnética/métodos , Desenho de PróteseRESUMO
Percutaneous mechanical circulatory support (MCS) devices are designed for short-term treatment in cases of acute decompensated heart failure as a bridge to transplant or recovery. Some of the known complications of MCS treatments are related to their hemodynamics in the aorta. The current study investigates the effect of MCS on the aortic flow. The study uses combined experimental and numerical methods to delineate complex flow structures. Particle image velocimetry (PIV) is used to capture the vortical and turbulent flow characteristics in a glass model of the human aorta. Computational fluid dynamics (CFD) analyses are used to complete the 3D flow in the aorta. Three specific MCS configurations are examined: a suction pump with a counterclockwise (CCW) rotating impeller, a suction pump with a clockwise (CW) rotating impeller, and a discharge pump with a straight jet. These models were examined under varying flow rates (1-2.5 L/min). The results show that the pump configuration strongly influences the flow in the thoracic aorta. The rotating impeller of the suction pump induces a dominant swirling flow in the aorta. The swirling flow distributes the incoming jet and reduces the turbulent intensity near the aortic valve and in the aorta. In addition, at high flow rates, the local vortices formed near the pump are washed downstream toward the aortic arch. Specifically, an MCS device with a CCW rotating impeller induces a non-physiological CCW helical flow in the descending aorta (which is opposite to the natural helical flow), while CW swirl combines better with the natural helical flow.
RESUMO
The present work investigates the combined effects of the upstream sediment mining pit and vegetation on the riverbank using emergent rigid vegetation beyond the toe on the flow structure and morphological changes due to fluvial erosion. A steep gradient of streamwise velocity and other turbulence parameters such as Reynolds shear stress (RSS), transverse RSS, and turbulent kinetic energy (TKE) at the interface of the vegetated and unvegetated part of the test segment was observed. The cross-sectional analysis showed that vegetation increased the velocity of the unvegetated main channel, and the sandpit increased even the near-bed velocity with a similar trend in its longitudinal variation at the center line of the main channel. The abrupt variation in RSS and transverse RSS at the location of the berm induces instability and erodes the berm present at the toe of the riverbank. The combination of the vegetation and sandpit led to increased TKE of the flow at the near-bed and berm locations. The morphological analysis showed complete riverbank erosion in both cases of the unvegetated riverbank, i.e., without or with an upstream pit. The installed stems of rigid vegetation on the riverbank helped decrease the fluvial erosion of the riverbank, and its profile observed minimal changes over the length of the test segment. However, the main channel erosion was amplified due to the vegetation (in no-pit case) at the beginning of the test segment, which eroded the bed of the main channel by about 67% of the bed thickness. Also, in the vegetated riverbank cases, the upstream pit caused an increase in erosion by 7.66% at the center of the main channel. The study helps establish the hypothesis of negating the effects of sediment mining on bank erosion by using the rigid vegetation on the riverbank beyond its toe location, which performed well by maintaining the riverbank profile.
RESUMO
Wind is an emerging renewable energy resource, but more useful in cold regions. With the increasing threat of climate change and global warming, the unpredictability of wind energy patterns has been affected. With continual threats from extremes and uncertainties, icing on wind turbines has been noted to grow affecting aerodynamic performance. The effect of turbulence intensity at its impact on aerodynamic performance was numerically done using ANSYS Fluent and FENSAP ICE software. Conditions considered for the study included turbulence intensities, median volume diameter (MVD), liquid water content (LWC), angle of attack, and ambient temperature for 180 min. The study's conditions aimed at providing a wide range of effects covering the in-cloud icing and freezing drizzle. The mass of ice increased with an increase in LWC when it increased from 0.05 g/m3 to 0.3 g/m3, and MVD with 1000 µm compared to 40 µm, but when temperature decreased led to an increase from -1 °C to -15 °C. Increasing the angle of attack led to reduced aerodynamic performance with stall angle occurring at α = 0-18°. An increase in the turbulence intensity from 0.01 % to 50 % resulted in decreased CL/CD.
RESUMO
Mpp46Ab is a mosquito-larvicidal pore-forming toxin derived from Bacillus thuringiensis TK-E6. Pore formation is believed to be a central mode of Mpp46Ab action, and the cation selectivity of the channel pores, in particular, is closely related to its mosquito-larvicidal activity. In the present study, we constructed a mutant library in which residue K155 within the transmembrane ß-hairpin was randomly replaced with other amino acid residues. Upon mutagenesis and following primary screening using Culex pipiens mosquito larvae, we obtained 15 mutants in addition to the wild-type toxin. Bioassays using purified proteins revealed that two mutants, K155E and K155I, exhibited toxicity significantly higher than that of the wild-type toxin. Although increased cation selectivity was previously reported for K155E channel pores, we demonstrated in the present study that the cation selectivity of K155I channel pores was also significantly increased. Considering the characteristics of the amino acids, the charge of residue 155 may not directly affect the cation selectivity of Mpp46Ab channel pores. Replacement of K155 with glutamic acid or isoleucine may induce a similar conformational change in the region associated with the ion selectivity of the Mpp46Ab channel pores. Mutagenesis targeting the transmembrane ß-hairpin may be an effective strategy for enhancing the ion permeability of the channel pores and the resulting mosquito-larvicidal activity of Mpp46Ab.
RESUMO
Turbulent blood flow is implicated in the pathogenesis of several aortic diseases but the extent and degree of turbulent blood flow in the normal aorta is unknown. We aimed to quantify the extent and degree of turbulece in the normal aorta and to assess whether age impacts the degree of turbulence. 22 young normal males (23.7 ± 3.0 y.o.) and 20 old normal males (70.9 ± 3.5 y.o.) were examined using four dimensional flow magnetic resonance imaging (4D Flow MRI) to quantify the turbulent kinetic energy (TKE), a measure of the intensity of turbulence, in the aorta. All healthy subjects developed turbulent flow in the aorta, with total TKE of 3-19 mJ. The overall degree of turbulence in the entire aorta was similar between the groups, although the old subjects had about 73% more total TKE in the ascending aorta compared to the young subjects (young = 3.7 ± 1.8 mJ, old = 6.4 ± 2.4 mJ, p < 0.001). This increase in ascending aorta TKE in old subjects was associated with age-related dilation of the ascending aorta which increases the volume available for turbulence development. Conversely, age-related dilation of the descending and abdominal aorta decreased the average flow velocity and suppressed the development of turbulence. In conclusion, turbulent blood flow develops in the aorta of normal subjects and is impacted by age-related geometric changes. Non-invasive assessment enables the determination of normal levels of turbulent flow in the aorta which is a prerequisite for understanding the role of turbulence in the pathophysiology of cardiovascular disease.
RESUMO
Cry46Ab is a Cry toxin derived from Bacillus thuringiensis TK-E6. Cry46Ab is not significantly homologous to other mosquitocidal Cry or Cyt toxins and is classified as an aerolysin-type pore-forming toxin based on structural similarity. In this study, the potency of Cry46Ab was assessed for its potential application to mosquito control. A synthetic Cry46Ab gene, cry46Ab-S1, was designed to produce recombinant Cry46Ab as a glutathione-S-transferase fusion in Escherichia coli. Recombinant Cry46Ab showed apparent toxicity to Culex pipiens larvae, with a 50% lethal dose of 1.02 µg/ml. In an artificial lipid bilayer, Cry46Ab activated by trypsin caused typical current transitions between open and closed states, suggesting it functions as a pore-forming toxin similar to other Cry and Cyt toxins. The single-channel conductance was 103.3 ± 4.1 pS in 150 mM KCl. Co-administration of recombinant Cry46Ab with other mosquitocidal Cry toxins, especially the combination of Cry4Aa and Cry46Ab, resulted in significant synergistic toxicity against C. pipiens larvae. Co-administration of multiple toxins exhibiting different modes of action is believed to prevent the onset of resistance in insects. Our data, taken in consideration with the differences in its structure, suggest that Cry46Ab could be useful in not only reducing resistance levels but also improving the insecticidal activity of Bt-based bio-insecticides.
Assuntos
Bacillus thuringiensis/genética , Toxinas Bacterianas/farmacologia , Culex , Proteínas Citotóxicas Formadoras de Poros , Animais , Toxinas Bacterianas/genética , Escherichia coli/genética , Larva , Controle de Mosquitos , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Recombinantes/genéticaRESUMO
Thermal gradient of a Central Amazonian lake was studied to establish a link between seasonal variations in the water level, temperature, suspended particulate matter (SPM) and thermal stratification. Bimonthly measurements of temperature and PAR radiation were made at 0.25 m intervals from the surface to bottom from February 2004 to July 2006. Daily occurs full vertical mixing of the water column, and classic thermal stratification was not observed in the period. The effect of the winds associated to flood-pulse and the penetrative convection, transported the turbulent kinetic energy (TKE) into the lake mixing the column of water. This phenomenon was more expressive in the rainfall seasons when is generally higher contributing to the circulation of the water. The limit of the euphotic zone ranged from 1.36 to 1.77 m in the period studied. The results of the transmission curves for the sampling sites showed that, in general, less than 0.01% of the surface light reached the bottom. The trend curve developed can facilitate the understanding of the limnological and ecological processes in lentic systems of whitewaters of the Central Amazonian.
Foi estudado o gradiente térmico de um lago da Amazônia Central para estabelecer associação entre variações sazonais no nível de água, temperatura, material em suspensão e estratificação térmica. Bimestralmente, foram medidas a temperatura e a radiação luminosa da superfície ao fundo do lago no período entre fevereiro de 2004 e julho de 2006. Ocorreu completa mistura vertical diária da coluna de água, não sendo observada estratificação térmica clássica no período. A energia cinética turbulenta (ECT) proveniente da ação dos ventos em associação com o pulso de inundação foi responsável pela mistura da coluna de água. Esse fenômeno foi mais expressivo no período chuvoso, quando a ECT é geralmente mais alta, contribuindo para a circulação da água. A extensão da zona eufótica variou de 1,36 a 1,77 m. Os resultados das curvas de transmissão para os pontos de amostragem mostraram que menos de 0,01% da luz de superfície alcançou o fundo do lago. A curva de tendência desenvolvida poderá facilitar a compreensão dos processos limnológicos e ecológicos em sistemas lênticos de águas brancas da Amazônia Central.