RESUMEN
Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.
Asunto(s)
Transporte Activo de Núcleo Celular , Señales de Localización Nuclear , Proteína FUS de Unión a ARN/química , beta Carioferinas/química , Sitios de Unión , Degeneración Lobar Frontotemporal/metabolismo , Humanos , Carioferinas/metabolismo , Luz , Extracción Líquido-Líquido , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética , Mutación , Nefelometría y Turbidimetría , Unión Proteica , Dominios Proteicos , ARN/química , Dispersión de Radiación , TemperaturaRESUMEN
To our knowledge, calibration curves or other validations for thousands of SomaScan aptamers are not publicly available. Moreover, the abundance of urine proteins obtained from these assays is not routinely validated with orthogonal methods (OMs). We report an in-depth comparison of SomaScan readout for 23 proteins in urine samples from patients with diabetic kidney disease (n = 118) vs OMs, including liquid chromatography-targeted mass spectrometry (LC-MS), ELISA, and nephelometry. Pearson correlation between urine abundance of the 23 proteins from SomaScan 3.2 vs OMs ranged from -0.58 to 0.86, with a median (interquartile ratio, [IQR]) of 0.49 (0.18, 0.53). In multivariable linear regression, the SomaScan readout for 6 of the 23 examined proteins (26%) was most strongly associated with the OM-derived abundance of the same (target) protein. For 3 of 23 (13%), the SomaScan and OM-derived abundance of each protein were significantly associated, but the SomaScan readout was more strongly associated with OM-derived abundance of one or more "off-target" proteins. For the remaining 14 proteins (61%), the SomaScan readouts were not significantly associated with the OM-derived abundance of the targeted proteins. In 6 of the latest group, the SomaScan readout was not associated with urine abundance of any of the 23 quantified proteins. To sum, over half of the SomaScan results could not be confirmed by independent orthogonal methods.
Asunto(s)
Nefropatías Diabéticas , Humanos , Nefropatías Diabéticas/orina , Cromatografía Liquida/métodos , Masculino , Femenino , Persona de Mediana Edad , Ensayo de Inmunoadsorción Enzimática , Proteómica/métodos , Espectrometría de Masas/métodos , Anciano , Nefelometría y Turbidimetría , Biomarcadores/orina , Proteinuria/orinaRESUMEN
Fitness in micro-organisms can be proxied by growth parameters on different media and/or temperatures. This is achieved by measuring optical density at 600 nm using a spectrophotometer, which measures the effect of absorbance and side scattering due to turbidity of cells suspensions. However, when growth kinetics must be monitored in many 96-well plates at the same time, buying several 96-channel spectrophotometers is often beyond budgets. The MiniRead device presented here is a simple and inexpensive do-it-yourself 96-well temperature-controlled turbidimeter designed to measure the interception of white light via absorption or side scattering through liquid culture medium. Turbidity is automatically recorded in each well at regular time intervals for up to several days or weeks. Output tabulated text files are recorded into a micro-SD memory card to be easily transferred to a computer. We propose also an R package which allows (1) to compute the nonlinear calibration curves required to convert raw readings into cell concentration values, and (2) to analyze growth kinetics output files to automatically estimate proxies of growth parameters such as lag time, maximum growth rate, or cell concentration at the plateau.
Asunto(s)
Nefelometría y Turbidimetría , Cinética , Nefelometría y Turbidimetría/instrumentación , Medios de Cultivo/química , Espectrofotometría/instrumentaciónRESUMEN
BACKGROUND: Detection of IgG subclasses (IgGSc) is vital for the diagnosis and management of disease, especially IgG4-related diseases (IgG4-RD). This study aimed to evaluate the performances of the chemiluminescent immunoassay (CLIA) for detecting IgGSc and diagnosing IgG4-RD by IgGSc. METHODS: A total of 40 individuals with IgG4-RD, 40 with primary Sjogren's syndrome (pSS), and 40 healthy controls (HCs) were enrolled. Serum samples were collected for the simultaneous detection of IgG1, IgG2, IgG3, and IgG4 by the Siemens immunonephelometric assay and the CLIA. The correlation analysis was performed, and diagnostic value was analyzed by the receiver operating characteristic (ROC) curve. RESULTS: Patients with IgG4-RD had higher IgG4 (p < 0.001) and lower IgG1 (p < 0.001) than those with pSS, and HC. The results by the Siemens immunonephelometric assay and the CLIA showed a strong correlation in detecting IgG1, IgG2, IgG3, and IgG4 (r = 0.937, r = 0.847, r = 0.871, r = 0.990, all p < 0.001, respectively). The sum of IgG1, IgG2, IgG3, and IgG4 using two assays strongly correlated with total IgG by the IMMAGE 800 (r = 0.866, r = 0.811, both p < 0.001, respectively). For discriminating IgG4-RD from pSS and HC, no significant differences were observed in CLIA IgG4 and Siemens immunonephelometric assay IgG4 (z = 0.138, p = 0.891), which provided the area under the curves (AUCs) of 0.951 (p < 0.001) and 0.950 (p < 0.001), respectively. The AUCs of CLIA IgG1 and Siemens immunonephelometric assay IgG1 in distinguishing pSS from IgG4-RD and HC were 0.761 (p < 0.001) and 0.765 (p < 0.001), respectively, with no significant differences (z = 0.228, p = 0.820). CONCLUSIONS: The CLIA and the Siemens immunonephelometric assay appeared to have good consistency with comparable diagnostic value in detecting IgGSc, especially IgG4, and IgG1 that can accurately identify IgG4-RD or pSS in clinical practice.
Asunto(s)
Inmunoglobulina G , Mediciones Luminiscentes , Humanos , Inmunoglobulina G/sangre , Femenino , Masculino , Persona de Mediana Edad , Inmunoensayo/métodos , Mediciones Luminiscentes/métodos , Adulto , Curva ROC , Nefelometría y Turbidimetría/métodos , Estudios de Casos y Controles , China , Anciano , Síndrome de Sjögren/sangre , Síndrome de Sjögren/diagnóstico , Pueblo Asiatico , Enfermedad Relacionada con Inmunoglobulina G4/sangre , Enfermedad Relacionada con Inmunoglobulina G4/diagnóstico , Pueblos del Este de AsiaRESUMEN
Polarization imaging techniques have more prominent advantages for imaging in strongly scattered media. Previous de-scattering methods of polarization imaging usually require the priori information of the background region, and rarely consider the effect of non-uniformity of the optical field on image recovery, which not only reduces the processing speed of imaging but also introduces errors in image recovery, especially for moving targets in complex scattering environments. In this paper, we propose a turbid underwater moving image recovery method based on the global estimation of the intensity and the degree of polarization (DOP) of the backscattered light, combined with polarization-relation histogram processing techniques. The full spatial distribution of the intensity and the DOP of the backscattered light are obtained by using frequency domain analysis and filtering. Besides, a threshold factor is set in the frequency domain low-pass filter, which is used to adjust the execution region of the filter, which effectively reduces the error in image recovery caused by estimating the DOP of the backscattered light as a constant in traditional methods with non-uniform illumination. Meanwhile, our method requires no human-computer interaction, which effectively solves the drawbacks that the moving target is difficult to be recovered by traditional methods. Experimental studies were conducted on static and moving targets under turbid water, and satisfactory image recovery quality is achieved.
Asunto(s)
Diagnóstico por Imagen , Iluminación , Nefelometría y Turbidimetría , Dispersión de Radiación , Refracción OcularRESUMEN
This study aimed to examine the agreement of the serum amyloid A (SAA) values determined using the ELISA test and the nephelometric automated method. This study included 80 serum samples obtained from patients with COVID-19. Samples were determined using ELISA and the nephelometric method. Wilcoxon signed ranks test showed a statistically significant difference in the calculated median values (Z = -2.432, p = 0.015). The correlation between methods was statistically significant (r = 0.603, p < 0.0001). Bland Altman analysis showed a bias of 56.6 mg/L and a relative bias of 7.4% between the methods. The results of this study indicate that further studies are needed that will examine the compliance between the ELISA and the nephelometric method for determining SAA, and the results must be carefully interpreted based on the method used.
Asunto(s)
COVID-19 , Proteína Amiloide A Sérica , Humanos , Proteína Amiloide A Sérica/análisis , COVID-19/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Nefelometría y TurbidimetríaRESUMEN
We established an experimental apparatus by combining polarized light scattering and angle-resolved light scattering measurement technology to rapidly identify the shape of an individual aerosol particle. The experimental data of scattered light of Oleic acid, rod-shaped Silicon dioxide, and other particles with typical shape characteristics were analyzed statistically. To better study the relationship between the shape of particles and the properties of scattered light, the partial least squares discriminant analysis (PLS-DA) method was used to analyze the scattered light of aerosol samples based on the size screening of particles, and the shape recognition and classification method of the individual aerosol particle was established based on the analysis of the spectral data after nonlinear processing and grouping by particle size with the area under the receiver operating characteristic curve (AUC) as reference. The experimental results show that the proposed classification method has a good discrimination ability for spherical, rod-shaped, and other non-spherical particles, which can provide more information for atmospheric aerosol measurement, and has application value for traceability and exposure hazard assessment of aerosol particles.
Asunto(s)
Tamaño de la Partícula , Nefelometría y Turbidimetría , AerosolesRESUMEN
Nanoparticles have been used as antibacterial agents in several products. To optimize their effectiveness, synthesis processes and particle modifications have been developed, creating the need for a rapid screening method to investigate their potencies. Owing to the opacity and insolubility of nanoparticles, a classical method to determine antibacterial activity-such as the minimum inhibitory concentration (MIC), which relies on turbidimetry-might not apply to them. In this study, we demonstrate the potential of a dye (resazurin)-based assay as an indicator of bacterial growth to rapidly screen the antibacterial activities of both organic and inorganic nanomaterials against both gram-negative (E. coli) and gram-positive (S. aureus) bacteria. The results indicate that the resazurin-based assay successfully determine the MIC of organic lipid nanocarriers, and several inorganic nanoparticles. However, the use of resazurin require a precaution for nanoparticles with photocatalytic properties, which may cause dye degradation at higher concentrations. In this study, resazurin bleaching was observed at approximately >50 mg/ml of TiO2. In summary, the modified MIC assay with resazurin can evaluate antibacterial activity of nanomaterials, whose turbidity interferer conventional MIC assay. This modification conserves an advantage of MICs assay which are simple and reliable. This would be useful for screening of antibacterial nanomaterials.
Asunto(s)
Antibacterianos/administración & dosificación , Escherichia coli/efectos de los fármacos , Nanopartículas/administración & dosificación , Oxazinas/química , Staphylococcus aureus/efectos de los fármacos , Xantenos/química , Antibacterianos/química , Indicadores y Reactivos/química , Nanopartículas del Metal/administración & dosificación , Nanopartículas del Metal/química , Pruebas de Sensibilidad Microbiana/métodos , Nanopartículas/química , Nefelometría y Turbidimetría/métodos , Aceites Volátiles/química , Tamaño de la Partícula , Plata/química , Titanio/química , Óxido de Zinc/químicaRESUMEN
BACKGROUND: The purpose of this study was to investigate the immunological and physical characteristics of IgM-λ type M-protein from patients who were measured low in the turbidimetric immunoassay (TIA) IgM assay without error codes for high concentration to determine the cause of the false low levels and to clarify the mechanism of their occurrence. METHODS: Materials were IgM patient samples and 8 serum samples from other IgM M-protein patients as controls. Patient samples were assayed by the TIA method, in which five manufacturers and six models (two reagent manufacturers) share the principle, and the BN ProSpec method (nephelometric method), which has a different principle. Dilution linearity tests, IgG addition experiments, isoelectric point electrophoresis, and hydrophobic chromatography were performed on patients and subjects. In addition, the binding capacity of γ-globulin by BIACORE was also examined. RESULTS: The reaction curve of the patient IgM curved downward when the concentration of IgM exceeded 20 g/L, and no error code was obtained. In the measurement by the TIA method of five manufacturers and six models, patient IgM was measured at a false low level with no error code obtained in undiluted dilution by any of the instruments and reagents, but could be measured without any problem by the nephelometric method. In addition, in the patient IgG addition experiment, only patient IgM showed a false low level under high IgG concentration. Furthermore, the binding capacity of patient IgM to γ-globulin (IgG) by BIACORE was significantly higher than that of the control IgM-type M protein. CONCLUSIONS: Patient IgM has an affinity (binding capacity) for IgG and forms an IgM-IgG complex under conditions of high IgG concentration. It was speculated that this complex inhibited the reaction with the anti-IgM antibody and the absorbance of the second reaction did not increase, suggesting a false low.
Asunto(s)
Inmunoturbidimetría , gammaglobulinas , Humanos , Inmunoglobulina M , Nefelometría y Turbidimetría , Indicadores y Reactivos , Inmunoglobulina G , Inmunoensayo/métodosRESUMEN
The determination of the optical properties in turbid media plays an essential role in medical diagnostics and process control. The method of spatially resolved reflectance measurements is a frequently used tool to evaluate the reduced scattering coefficient as well as the absorption coefficient. In most cases a smooth interface is assumed between the medium under investigation and the surrounding medium. However, in reality, a rough surface is present at the interface, which alters the light interaction with the surface and volume of the turbid medium. Hence, the idea behind this paper was to investigate the influence of rough surfaces on the spatially resolved reflectance and thus on the determination of the optical properties of turbid media. Particularly, the influence of a Lambertian scattering surface on the result of Monte Carlo simulations of a spatially resolved reflectance setup is shown. In addition, we distinguish between the different interaction modes of surface scattering on the spatially resolved reflectance. There is a strong influence of roughness when the light enters and leaves the turbid medium. Furthermore, the simulations show that, especially for small reduced scattering coefficients and absorption coefficients, large errors in the determination of the optical properties are obtained.
Asunto(s)
Dispersión de Radiación , Método de Montecarlo , Nefelometría y TurbidimetríaRESUMEN
Spatially resolved reflectance measurements are a standard tool for determining the absorption and scattering properties of turbid media such as biological tissue. However, in literature, it was shown that these measurements are subject to errors when a possible rough surface between the turbid medium and the surrounding is not accounted for. We evaluated these errors by comparing the spatially resolved reflectance measured on rough epoxy-based samples with Monte Carlo simulations using Lambertian surface scattering, the Cook-Torrance model, and the generalized Harvey-Shack model as surface scattering models. To this aim, goniometric measurements on the epoxy-based samples were compared to the angularly resolved reflectance of the three surface models to estimate the corresponding model parameters. Finally, the optical properties of the phantoms were determined using a Monte Carlo model with a smooth surface.
Asunto(s)
Nefelometría y Turbidimetría , Dispersión de Radiación , Método de Montecarlo , Fantasmas de ImagenRESUMEN
The turbidity assay is commonly exploited to study protein liquid-to-liquid phase separation (LLPS) or liquid-to-solid phase separation (LSPS) processes in biochemical analyses. Herein, we present common pitfalls of this assay caused by exceeding the detection linear range. We showed that aggregated proteins of high concentration and large particle size can lead to inaccurate quantification in multiple applications, including the optical density measurement, the thermal shift assay, and the dynamic light scattering experiment. Finally, we demonstrated that a simple sample dilution of insoluble aggregated protein (LSPS) samples or direct imaging of liquid droplets (LLPS) can address these issues and improve the accuracy of the turbidity assay.
Asunto(s)
Fraccionamiento Químico/métodos , Nefelometría y Turbidimetría/métodos , Proteínas/química , Proteínas/aislamiento & purificación , Amiloide/análisis , Amiloide/química , Dispersión Dinámica de Luz , Cinética , Límite de Detección , Tamaño de la Partícula , Agregado de Proteínas , Análisis EspectralRESUMEN
Macromolecular crowding plays an inevitable role in all biological processes influencing association, conformation, and other characteristics of proteins. Present study is based on the effect of macromolecular crowding on structure of horseradish peroxidase (HRP) enzyme. Concentration-dependent conformational changes induced by crowding agents, dextran 70 and polyethylene glycol (PEG)-4000, were monitored employing a range of biophysical techniques. The intrinsic fluorescence spectra showed transition of protein from native to unfolded state. Marked increase in 8-Anilino-1-naphthalene-sulphonoic acid and Thioflavin T fluorescence indicated presence of non-native moieties with 80 mg/mL dextran. Enhanced absorbance in turbidity, Soret, and Congo red in corroboration with scattering intensity at 350nm results revealed incidence of HRP aggregates. A new peak around 218 nm in CD spectra pointed towards change in secondary structure towards ß-sheets. Significant loss of enzyme activity upon structural disruption was seen. Comet assay demonstrated DNA damage and genotoxic nature of HRP aggregates, supporting spectroscopic, and fluorescence results. The normalized results were obtained with 120 mg/mL PEG-4000 close to that of native HRP implying no disruptive effect on structure. It can be hypothesized that macromolecular crowding is a vital element, which can have diverse effects. In this study, dextran 70 was observed to have pro-aggregatory effect while enhanced stability of native enzyme was witnessed with PEG. Hence, it can be stated that PEG has potentially better crowder as it helps retain the native enzyme structure. Routine addition of crowding agents is recommended if biological molecules are to be studied under more physiologically appropriate environments.
Asunto(s)
Dextranos/química , Peroxidasa de Rábano Silvestre/química , Polietilenglicoles/química , Acrilamida/química , Benzotiazoles/química , Dicroismo Circular , Ensayo Cometa , Peroxidasa de Rábano Silvestre/metabolismo , Peroxidasa de Rábano Silvestre/farmacología , Humanos , Linfocitos , Nefelometría y Turbidimetría , Conformación Proteica en Lámina beta , Estabilidad Proteica , Estructura Secundaria de Proteína , Espectrometría de FluorescenciaRESUMEN
The presence of hydrosols, taken as suspension of micro- or macroscopic material in water, strongly alters light propagation and thus the radiance distribution within a natural or artificial water volume. Understanding of hydrosols' impacts on light propagation is limited by our ability to accurately handle the angular scattering phase function inherent to complex material such as suspended sediments or living cells. Based on actual quality-controlled measurements of sediments and microalgae, this Letter demonstrates the superiority of a two-term five-parameter empirical phase function as recently proposed for scattering by nanoparticle layers [Nanoscale11, 7404 (2019)NANOHL2040-336410.1039/C9NR01707K]. The use of such phase function parameterizations presents new potentialities for various radiative transfer and remote sensing applications related to an aquatic environment.
Asunto(s)
Microalgas , Simulación por Computador , Luz , Nefelometría y Turbidimetría , Dispersión de RadiaciónRESUMEN
Many patients develop coagulation abnormalities due to chronic and hereditary disorders, infectious disease, blood loss, extracorporeal circulation, and oral anticoagulant misuse. These abnormalities lead to bleeding or thrombotic complications, the risk of which is assessed by coagulation analysis. Current coagulation tests pose safety concerns for neonates and small children due to large sample volume requirement and may be unreliable for patients with coagulopathy. This study introduces a containerless drop-of-blood method for coagulation analysis, termed "integrated quasi-static acoustic tweezing thromboelastometry" (i-QATT™), that addresses these needs. In i-QATT™, a single drop of blood is forced to levitate and deform by the acoustic radiation force. Coagulation-induced changes in drop turbidity and firmness are measured simultaneously at different instants. The parameters describing early, intermediate, and late stages of the coagulation process are evaluated from the resulting graphical outputs. i-QATT™ rapidly (<10 min) detected hyper- and hypo-coagulable states and identified single deficiency in coagulation factors VII, VIII, IX, X, and XIII. The linear relationship (r2 > 0.9) was established between fibrinogen concentration and two i-QATT™ parameters: maximum clot firmness and maximum fibrin level. Factor XIII activity was uniquely measured by the fibrin network formation time (r2 = 0.9). Reaction time, fibrin formation rate, and time to firm clot formation were linearly correlated with heparin concentration (r2 > 0.7). tPA-induced hyperfibrinolysis was detected in the clot firmness output at 10 min. i-QATT™ provides comprehensive coagulation analysis in point-of-care or laboratory settings, well suited to the needs of neonatal and pediatric patients and adult patients with anemia or blood collection issues.
Asunto(s)
Coagulación Sanguínea , Nefelometría y Turbidimetría/métodos , Tromboelastografía/métodos , Anticoagulantes/uso terapéutico , Monitoreo de Drogas , Estudios de Factibilidad , HumanosRESUMEN
Optical proxies based on light scattering measurements have potential to improve the study and monitoring of aquatic environments. In this study, we evaluated several optical proxies for characterization of particle mass concentration, composition, and size distribution of suspended particulate matter from two contrasting coastal marine environments. We expanded upon our previous study of Southern California coastal waters, which generally contained high proportions of organic particles, by conducting angle-resolved polarized light scattering measurements in predominantly turbid and inorganic-particle dominated Arctic coastal waters near Prudhoe Bay, Alaska. We observed that the particulate backscattering coefficient bbp was the most effective proxy for the mass concentration of suspended particulate matter (SPM) when compared with particulate scattering and attenuation coefficients bp and cp. Improvements were seen with bbp as a proxy for the concentration of particulate organic carbon (POC), although only if particulate assemblages were previously classified in terms of particle composition. We found that the ratio of polarized-light scattering measurements at 110º and 18º was superior in performance as a proxy for the composition parameter POC/SPM in comparison to the particulate backscattering ratio bbp/bp. The maximum value of the degree of linear polarization DoLPp,max observed within the range of scattering angles 89°-106° was found to provide a reasonably good proxy for a particle size parameter (i.e., 90th percentile of particle volume distribution) which characterizes the proportions of small- and large-sized particles. These findings can inform the development of polarized light scattering sensors to enhance the capabilities of autonomous platforms.
Asunto(s)
Material Particulado , Nefelometría y Turbidimetría , Isótopos de Oxígeno , Material Particulado/análisis , Dispersión de RadiaciónRESUMEN
INTRODUCTION: The extended stability of the trastuzumab biosimilar Ogivri™ (MYL-1401O; trastuzumab-dkst) was studied under different storage conditions, including following reconstitution of the lyophilized powder (21 mg/mL) but undiluted and stored in vials at 4°C; after dilution at two concentrations (0.8 and 2.4 mg/mL) in polyolefin bags and stored at 4°C; and following a three-day thermal excursion to 25°C. METHODS: Several methods were utilized to assess the physical and chemical stability of the drug under different storage conditions. RESULTS: At all storage conditions tested, there was no change in the tertiary structure of MYL-1401O as assessed by second-derivative ultraviolet and fluorescence-derived spectral analysis, and no evidence of oligomer formation or fragmentation was observed as assessed by gel exclusion chromatography and dynamic light scattering, confirmed by assessment of quinary structures using size-exclusion chromatography. Ion-exchange chromatography showed no significant changes in the distribution of ionic variants, particularly deamidations. Thermal denaturation curves indicated no destabilization of the three-dimensional structure after 90 days at 4°C or after thermal excursion for 72 h at 25°C. CONCLUSION: The trastuzumab biosimilar MYL-1401O maintained its physical and chemical stability for at least 90 days at 4°C or after thermal excursion to 25°C, supporting the safe use of MYL-1401O in several real-world settings, including advanced preparation for administration or when a break in the cold cycle occurs.
Asunto(s)
Biosimilares Farmacéuticos/química , Filgrastim/química , Polietilenglicoles/química , Trastuzumab/química , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Embalaje de Medicamentos , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Luz , Nefelometría y Turbidimetría , Polvos , Dispersión de Radiación , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
It is widely acknowledged that faster-growing bacteria are killed faster by ß-lactam antibiotics. This notion serves as the foundation for the concept of bacterial persistence: dormant bacterial cells that do not grow are phenotypically tolerant against ß-lactam treatment. Such correlation has often been invoked in the mathematical modeling of bacterial responses to antibiotics. Due to the lack of thorough quantification, however, it is unclear whether and to what extent the bacterial growth rate can predict the lysis rate upon ß-lactam treatment under diverse conditions. Enabled by experimental automation, here we measured >1,000 growth/killing curves for eight combinations of antibiotics and bacterial species and strains, including clinical isolates of bacterial pathogens. We found that the lysis rate of a bacterial population linearly depends on the instantaneous growth rate of the population, regardless of how the latter is modulated. We further demonstrate that this predictive power at the population level can be explained by accounting for bacterial responses to the antibiotic treatment by single cells. This linear dependence of the lysis rate on the growth rate represents a dynamic signature associated with each bacterium-antibiotic pair and serves as the quantitative foundation for designing combination antibiotic therapy and predicting the population-structure change in a population with mixed phenotypes.
Asunto(s)
Antibacterianos/farmacología , Bacteriólisis/efectos de los fármacos , Carbenicilina/farmacología , Escherichia coli/efectos de los fármacos , Carga Bacteriana , Biomasa , Medios de Cultivo/farmacología , Escherichia coli/crecimiento & desarrollo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Cinética , Nefelometría y Turbidimetría , Robótica , TemperaturaRESUMEN
The diagnosis, prognosis, and control of chronic kidney disease rely on an understanding of the glomerular filtration rate (GFR). The renal clearance of the cystatin-C is closely associated with the GFR. Cystatin-C is a more suitable GFR marker than the commonly used creatinine. General techniques for cystatin-C calculation, such as particle-enhanced turbidimetric and nephelometric assay, are time-consuming and tedious. Here, we propose a rapid, quantitative immunoassay for the detection of cystatin-C. A fluorescence-based lateral-flow kit was developed in a sandwich format by using a monoclonal antibody. A Linear calibration was obtained over the clinical diagnostic range of 0.023-32 µg/mL and the limit of detection (LOD) was 0.023 µg/mL and the limit of quantification (LOQ) was 0.029 µg/mL. Average recoveries from spiked urine samples ranged from 96-100% and the coefficient of variation was less than 4% for both intra and inter-day assays with excellent repeatability. With the comparison with an ELISA kit, the developed kit is highly sensitive, performs well over the detection range, provides repeatable results in a short time, and can easily be used at point-of-care (POC), making it an ideal candidate for rapid testing in early detection, community screening for renal function disorders.
Asunto(s)
Insuficiencia Renal Crónica , Biomarcadores , Creatinina , Tasa de Filtración Glomerular , Humanos , Inmunoensayo , Nefelometría y Turbidimetría , Insuficiencia Renal Crónica/diagnósticoRESUMEN
Several degenerative amyloid diseases, with no fully effective treatment, affect millions of people worldwide. These pathologies-amyloidoses-are known to be associated with the formation of ordered protein aggregates and highly stable and insoluble amyloid fibrils, which are deposited in multiple tissues and organs. The disruption of preformed amyloid aggregates and fibrils is one possible therapeutic strategy against amyloidosis; however, only a few compounds have been identified as possible fibril disruptors in vivo to date. To properly identify chemical compounds as potential fibril disruptors, a reliable, fast, and economic screening protocol must be developed. For this purpose, three amyloid fibril formation protocols using transthyretin (TTR), a plasma protein involved in several amyloidoses, were studied using thioflavin-T fluorescence assays, circular dichroism (CD), turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM), in order to characterize and select the most appropriate fibril formation protocol. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was successfully used to study the interaction of doxycycline, a known amyloid fibril disruptor, with preformed wild-type TTR (TTRwt) aggregates and fibrils. DLS and TEM were also used to characterize the effect of doxycycline on TTRwt amyloid species disaggregation. A comparison of the TTR amyloid morphology formed in different experimental conditions is also presented.