RESUMO
In recent years, the use of prescribed and non-prescribed drugs has increased. Therefore, advances in new technologies and sensors for detecting molecules in natural environments are required. In this work, a 3D-printed polylactic acid stencil is used to fabricate paper-based analytical devices (ePADs). Herein, we report the use of carbon-based lab-manufactured conductive ink for the fabrication of sensors towards the detection of chloroquine and escitalopram. For each batch, eight ePADs were successfully fabricated. Firstly, the fabricated sensors were evaluated morphologically by scanning electron microscopy and electrochemically by cyclic voltammetry and electrochemical impedance spectroscopy experiments. The sensors displayed a well-defined voltammetric profile in the presence of the redox couple, when compared to a commercial carbon screen-printed electrode. Differential pulse voltammetry conducted the detection of chloroquine and escitalopram with detection limits of 4.0 and 0.5 µmol L-1, respectively. The ePADs fabricated using the 3D stencil are here presented as alternatives for the fabrication of electrochemical analytical devices. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10008-021-05075-w.
RESUMO
Hyperinsulinemia, which is associated with aging and metabolic disease, may lead to defective protein homeostasis (proteostasis) due to hyperactivation of insulin-sensitive pathways such as protein synthesis. We investigated the effect of chronic hyperinsulinemia on proteostasis by generating a time-resolved map of insulin-regulated protein turnover in adipocytes using metabolic pulse-chase labeling and high resolution mass spectrometry. Hyperinsulinemia increased the synthesis of nearly half of all detected proteins and did not affect protein degradation despite suppressing autophagy. Unexpectedly, this marked elevation in protein synthesis was accompanied by enhanced protein stability and folding and not by markers of proteostasis stress such as protein carbonylation and aggregation. The improvement in proteostasis was attributed to a coordinate up-regulation of proteins in the global proteostasis network, including ribosomal, proteasomal, chaperone, and endoplasmic reticulum/mitochondrial unfolded protein response proteins. We conclude that defects associated with hyperactivation of the insulin signaling pathway are unlikely attributed to defective proteostasis because up-regulation of protein synthesis by insulin is accompanied by up-regulation of proteostatic machinery.
Assuntos
Adipócitos/metabolismo , Insulina/metabolismo , Biossíntese de Proteínas , Carbonilação Proteica , Proteólise , Transdução de Sinais , Resposta a Proteínas não Dobradas , Células 3T3-L1 , Adipócitos/patologia , Animais , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patologia , CamundongosRESUMO
Rapidly cooling pigs after heat stress (HS) results in a pathophysiological condition, and because rapid temperature fluctuations may be associated with reduced reproductive success in sows, it lends itself to the hypothesis that these conditions may be linked. Objectives were to determine the effects of rapid cooling on thermal response and future reproductive success in pigs. Thirty-six replacement gilts (137.8±0.9kg BW) were estrus synchronized and then 14.1±0.4 d after estrus confirmation, pigs were exposed to thermoneutral conditions (TN; n=12; 19.7±0.9°C) for 6h, or HS (36.3±0.5°C) for 3h, followed by 3h of rapid cooling (HSRC; n=12; immediate TN exposure and water dousing) or gradual cooling (HSGC; n=12; gradual decrease to TN conditions) repeated over 2 d. Vaginal (TV) and gastrointestinal tract temperatures (TGI) were obtained every 15min, and blood was collected on d 1 and d 2 during the HS and recovery periods at 180 and 60min, respectively. Pigs were bred 8.3±0.8 d after thermal treatments over 2 d. Reproductive tracts were collected and total fetus number and viability were recorded 28.0±0.8 d after insemination. HS increased TV and TGI (P=0.01; 0.98°C) in HSRC and HSGC compared to TN pigs. During recovery, TV was reduced from 15 to 105min (P=0.01; 0.33°C) in HSRC compared to HSGC pigs, but no overall differences in TGI were detected (P<0.05; 39.67°C). Rapid cooling increased (P<0.05) TNFα compared to HSGC and TN pigs during recovery-d 1 (55.2%), HS-d 2 (35.1%), and recovery-d 2 (64.9%). Viable fetuses tended to be reduced (P=0.08; 10.5%) and moribund fetuses tended to be increased (P=0.09; 159.3%) in HSRC compared to HSGC and TN pigs. In summary, rapid cooling prior to breeding may contribute to reduced fetal viability and reproductive success in pigs.
Assuntos
Temperatura Corporal , Reprodução , Estresse Fisiológico , Suínos/fisiologia , Animais , Regulação da Temperatura Corporal , Cruzamento , Temperatura Baixa , Resposta ao Choque Frio , Feminino , Transtornos de Estresse por Calor/sangue , Transtornos de Estresse por Calor/fisiopatologia , Transtornos de Estresse por Calor/veterinária , Resposta ao Choque Térmico , Temperatura Alta , Resistência à Insulina , Masculino , Suínos/sangue , Fator de Necrose Tumoral alfa/sangueRESUMO
Over 70 species of intertidal fishes from 12 families breathe air while emerging from water. Amphibious intertidal fishes generally have no specialized air-breathing organ but rely on vascularized mucosae and cutaneous surfaces in air to exchange both oxygen and carbon dioxide. They differ from air-breathing freshwater fishes in morphology, physiology, ecology and behaviour. Air breathing and terrestrial activity are present to varying degrees in intertidal fish species, correlated with the tidal height of their habitat. The gradient of amphibious lifestyle includes passive remainers that stay in the intertidal zone as tides ebb, active emergers that deliberately leave water in response to poor aquatic conditions and highly mobile amphibious skipper fishes that may spend more time out of water than in it. Normal terrestrial activity is usually aerobic and metabolic rates in air and water are similar. Anaerobic metabolism may be employed during forced exercise or when exposed to aquatic hypoxia. Adaptations for amphibious life include reductions in gill surface area, increased reliance on the skin for respiration and ion exchange, high affinity of haemoglobin for oxygen and adjustments to ventilation and metabolism while in air. Intertidal fishes remain close to water and do not travel far terrestrially, and are unlikely to migrate or colonize new habitats at present, although in the past this may have happened. Many fish species spawn in the intertidal zone, including some that do not breathe air, as eggs and embryos that develop in the intertidal zone benefit from tidal air emergence. With air breathing, amphibious intertidal fishes survive in a variable habitat with minimal adjustments to existing structures. Closely related species in different microhabitats provide unique opportunities for comparative studies.
Assuntos
Peixes/fisiologia , Respiração , Fenômenos Fisiológicos da Pele , Adaptação Fisiológica , Aerobiose , Ar , Anaerobiose , Animais , Ecossistema , Peixes/metabolismo , Hipóxia , Oxigênio/metabolismo , Ondas de MaréRESUMO
Enzymatic electrochemical biosensors play an important role in the agri-food sector due to the need to develop sustainable, low-cost, and easy-to-use analytical devices. Such biosensors can be used to monitor pathogens, endocrine disruptors, and pesticides, such as carbaryl, widely used in many crops. The use of renewable carbon (RC) sources, provided from biomass pyrolysis has been often applied in the fabrication of such sensors. This material is a great candidate for biosensor fabrication due to the presence of surface functional groups, porosity, and moderate surface area. This work describes the functionalization of RC material through an acid treatment with a sulfonitric solution HNO3/H2SO4 (1:3) and the resulting material was characterized by scanning electron microscopy. The obtained RC functionalized (RCF) and the acetylcholinesterase enzyme (AChE) were applied in the construction of the electrochemical biosensor on glassy carbon (GC) electrode and used to detect carbaryl in apple samples. The GC/RCF/AChE biosensor was able to detect the carbaryl pesticide from 5.0 to 30.0 nmol L-1, displaying a LOD of 4.5 nmol L-1. The detection of carbaryl in apple samples presented recoveries between 102.5 to 118.6% through the standard addition method. The proposed biosensor is a promising renewable tool for food safety.
Assuntos
Técnicas Biossensoriais , Praguicidas , Acetilcolinesterase/química , Técnicas Biossensoriais/métodos , Carbaril , Carbono/química , Enzimas Imobilizadas/químicaRESUMO
Due to the SARS-CoV-2 pandemic, there has been an increase in the search for affordable healthcare devices for mass testing and rapid diagnosis. In this context, this work described a new methodology for SARS-CoV-2 detection based on an impedimetric immunosensor developed using the advantageous immobilization of antibodies in the reduced graphene oxide (rGO). The rGO was obtained by chemical synthesis from the commercial graphene oxide (GO), and the materials were morphologically, electrochemically and visually characterized. The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were used to evaluate the fabrication steps of the immunosensor. The electrochemical immunoassay was considered for SARS-CoV-2 spike protein RBD detection using a impedimetric immunosensor and redox couple ([(Fe(CN)6)]3-/4-) as a probe. The immunosensor was effectively developed and applied in the detection of SARS-CoV-2 spike protein RBD in saliva samples.
RESUMO
A simple, cheap, and less aggressive immobilization procedure for biomolecules using reduced graphene oxide (rGO) was employed to prepare an impedimetric immunosensor for detection of staphylococcal enterotoxin A (SEA) from Staphylococcus aureus in milk samples. The scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS) were used to monitor the single steps of the electrode assembly process. The glassy carbon (GC)/rGO platform detected the antigen-antibody binding procedures of SEA with concentrations of 0.5 to 3.5 mg L-1 via impedance changes in a low frequency range. The impedimetric immunosensor was successfully applied for the determination of SEA in milk samples.
RESUMO
California grunion Leuresthes tenuis synchronize spawning with tidal cycles, so the embryos incubate in a terrestrial environment, delay hatching until cued by a specific environmental trigger, and may extend incubation for up to an additional four weeks. These adaptations, however, do not appear to alter the morphology or sequence of early developmental stages as compared to other Atherinomorph fishes in the Orders Beloniformes and Cyprinodontiformes. Embryonic development is described in a series of 30 stages based on morphology observed by light microscopy. Stages are placed in five periods: zygote and cleavage, blastula, gastrula, segmentation and organogenesis, and hatching competence. Embryos from a southern population of L. tenuis in Los Angeles are compared with embryos found > 560 km north in San Francisco Bay. Northern L. tenuis embryos developed more slowly at several stages than southern embryos and reached hatching competence later, but both locations maintained synchrony with the tidal cycle for both spawning and hatching. The variation in rates of development and stage at hatching readiness are forms of developmental heterochrony that may be associated with evolutionary adaptation or morphological plasticity within this highly successful clade.
Assuntos
Desenvolvimento Embrionário , Smegmamorpha/embriologia , Animais , Praias , Embrião não Mamífero/embriologia , Organogênese , Óvulo/citologia , Fatores de Tempo , Zigoto/citologiaRESUMO
Functional single nucleotide polymorphisms (SNPs) of interleukin (IL)-4 -590 (C>T), toll-like receptor (TLR)-2 +2258 (G>A) and matrix metalloproteinase (MMP)-9 -1562 (C>T) were examined by polymerase chain reaction-restriction fragment length polymorphism to identify their merit as genetic markers for pre-eclampsia. One hundred and seventeen pre-eclamptic women and 146 control subjects with uncomplicated singleton pregnancies participated in this study, conducted at Leeds General Infirmary and St James's University Hospital. While the TLR-2 +2258 (G>A) and MMP-9 -1562 (C>T) SNPs failed to present any significant association with pre-eclampsia, there was a marked trend for an association between the IL-4 -590 (C>T) SNP and pre-eclampsia (chi(2)= 5.87, P = 0.055), with a prevalence of TT homozygous women in this group (OR 4.455, 95% CI 1.286-15.350).
Assuntos
Interleucina-4/genética , Metaloproteinase 9 da Matriz/genética , Polimorfismo Genético/genética , Pré-Eclâmpsia/genética , Receptor 2 Toll-Like/genética , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Marcadores Genéticos , Predisposição Genética para Doença/genética , Humanos , GravidezRESUMO
BACKGROUND: Whether BRCA1 and BRCA2 mutation carriers have a clinically relevant elevated risk of uterine cancer has implications for risk-reducing surgery. AIM: This multicentre, prospective cohort study assessed uterine cancer risk for mutation carriers compared with the general population. METHODS: Eligible mutation carriers were enrolled in the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) cohort study, had a uterus present and no history of uterine cancer at cohort entry. Epidemiological, lifestyle and clinical data were collected at cohort entry and updated three-yearly. Cancer events were verified using pathology reports. Follow-up was censored at death or last contact. Relative risk of uterine cancer was estimated using the standardised incidence ratio (SIR), with the expected number of cases determined using population-based data for Australia. RESULTS: Of 1,111 mutation carriers in kConFab, 283 were excluded due to prior hysterectomy (N = 278), prior uterine cancer (N = 2) or being non-residents (N = 3). After a median follow-up of 9.0 years, five incident uterine cancers were reported in the 828 eligible women (419 had prior breast cancer and 160 had prior tamoxifen use), compared to 2.04 expected (SIR = 2.45; 95% confidence interval [CI]: 0.80-5.72; P = 0.11). In 438 BRCA1 mutation carriers and 390 BRCA2 mutation carriers, three and two incident cases of uterine cancer were reported, respectively, compared to 1.04 expected (SIR = 2.87; 95% CI: 0.59-8.43; P = 0.18) and 0.99 expected (SIR = 2.01; 95% CI: 0.24-7.30; P = 0.52), respectively. All cases were endometrioid subtype, International Federation of Gynaecology and Obstetrics stage I-II disease. No serous uterine cancers were reported. CONCLUSIONS: Our findings are consistent with those from most other reports and do not support routine risk-reducing hysterectomy for BRCA1 and BRCA2 mutation carriers.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Biomarcadores Tumorais/genética , Heterozigoto , Mutação , Neoplasias Uterinas/genética , Adulto , Austrália/epidemiologia , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Humanos , Histerectomia , Incidência , Pessoa de Meia-Idade , Nova Zelândia/epidemiologia , Fenótipo , Estudos Prospectivos , Fatores de Proteção , Medição de Risco , Fatores de Risco , Comportamento de Redução do Risco , Fatores de Tempo , Neoplasias Uterinas/diagnóstico , Neoplasias Uterinas/epidemiologia , Neoplasias Uterinas/prevenção & controleRESUMO
BACKGROUND: Disruptions to intermyocyte coupling have been implicated in arrhythmogenesis and development of conduction disturbances. At present, understanding of the relationship between the microscopic organization of intercellular coupling and the macroscopic spread of impulse in the normal and diseased heart is largely confined to theoretical analyses. METHODS AND RESULTS: The abundance and arrangement of gap junctions, as well as conduction properties, were assessed in terminal crest preparations isolated from the atria of neonate, weanling, and adult rabbits. We report that the connexin composition of terminal crest was uncomplicated, with Cx43 being the most prominent isoform detectable by Western blotting and immunostaining. Terminal crest myocytes showed little change in total Cx43-gap junction per cell during postnatal growth as assessed by stereology. However, marked non-uniformities emerged in the sarcolemmal distribution of Cx43-gap junctions. Cx43-gap junction area at myocyte termini increased 3.5-fold from birth to adulthood. Correlated with this change in Cx43, impulse propagation velocity parallel to the myofiber axis, as assessed by multi-site optical mapping using voltage-sensitive dye (di-4-ANEPPS), increased 2.4-fold. Conversely, the amount of Cx43-gap junctions on myocyte sides, and the conduction velocity transverse to the myofiber axis, remained relatively invariant during maturation. Hence, the increasing electrical anisotropy of maturing terminal crest was wholly accounted for by increases in conductance velocity along the bundle. This increase in longitudinal conduction velocity was correlated with changes in the sarcolemmal pattern, but not the overall density, of Cx43-gap junctions. CONCLUSIONS: This study provides the first correlative structure/function analysis of the relationship between the macroscopic conduction of impulse and the microscopic cellular organization of gap junctions in a differentiating cardiac bundle. Confirmation is provided for theoretical predictions which emphasize the importance of the cell-to-cell geometry of coupling in determining the spread and pattern of myocardial activation.
Assuntos
Conexina 43/análise , Junções Comunicantes/química , Coração/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Western Blotting , Conexinas/análise , Coração/crescimento & desenvolvimento , Sistema de Condução Cardíaco/fisiologia , Imuno-Histoquímica , Coelhos , Desmame , Proteína alfa-5 de Junções ComunicantesRESUMO
To define the changes in adrenal gland function during critical illness, we evaluated 28 severely ill patients with persistent hypotension who were hospitalized in a medical intensive care unit. The patients had increased plasma cortisol (mean +/- SE, 40.1 +/- 10.1 micrograms/dl). PRA was increased in all subjects (21.6 +/- 7.2 ng/ml.h); however, the plasma aldosterone concentration was inappropriately low in 18 of the subjects, with values ranging from 1-9 ng/dl, despite normal serum potassium concentrations (4.3 +/- 0.1 meq/liter) and increased concentrations of the aldosterone percursor, 18-hydroxycorticosterone. These 18 patients had hypotension associated with major infections and a high mortality rate (78%). Infusions of ACTH or angiotensin II were associated with a normal aldosterone response in only 2 of the 14 patients tested, also suggesting that the defect was probably at the level of the zone glomerulosa cell. Although infection was a common underlying illness, no other factors, such as dopamine administration, decreased angiotensin-converting enzyme activity, or increased aldosterone clearance, could be implicated as the cause of the phenomena. Thus, selective hypoaldosteronism in the presence of high renin levels exists in a substantial percentage of hypotensive critically ill patients.
Assuntos
Aldosterona/sangue , Renina/sangue , Doença Aguda , Glândulas Suprarrenais/patologia , Hormônio Adrenocorticotrópico/farmacologia , Adulto , Idoso , Angiotensina II/metabolismo , Eletrólitos/metabolismo , Feminino , Humanos , Hidrocortisona/farmacologia , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-IdadeRESUMO
A flow cytometer was used to measure the fluorescence intensity of Bacillus anthracis spores, B. subtilis spores and Escherichia coli stained in suspension with specific rabbit fluorescein-conjugated antibody. The effect of normal sera and a number of other additives on the binding of conjugate to the surface of the homologous bacteria was assessed by measuring the median fluorescence intensity of the bacterial population in the reaction mixture. Non-ionic detergent depressed binding of one conjugate (anti-E. coli) by up to 22%. Bovine serum albumin, gelatin, foetal calf serum and normal rabbit serum did not affect the median fluorescence value for these 3 bacterial species by more than 14%. Normal serum from 5 goats reduced the specific staining of B. anthracis by up to two-thirds. Anti-B. anthracis antibodies were detected in goat serum by indirect immunofluorescence microscopy, and it is inferred that these goat antibodies were in competition with fluorescein conjugate for the bacterial antigens. Normal goat and sheep serum stimulated the specific staining of B. subtilis and E. coli measured by the cytometer; in the case of goat serum previous heating of the serum to 56 degrees C resulted in repression of staining of E. coli. Since anti-E. coli antibody was detected in this normal sera by indirect immunofluorescence assays, it is proposed that repression was caused by anti-bacterial antibodies and stimulation by a separate factor, heat-labile in the case of goat serum. The stimulatory factor was also apparently inactivated by increasing the NaCl concentration, suggesting that stimulation depends heavily on charge interactions. Preliminary evidence is presented that the stimulatory factor may be anti-antibody, possibly of the IgA or IgG class.
Assuntos
Bacillus anthracis/análise , Bacillus subtilis/análise , Escherichia coli/análise , Citometria de Fluxo , Imunofluorescência , Esporos Bacterianos/análise , Animais , Bacillus anthracis/fisiologia , Fenômenos Fisiológicos Sanguíneos , Soluções Tampão/farmacologia , Bovinos , Reações Falso-Negativas , Cabras , OvinosRESUMO
A microfluorometer was constructed by modifying a standard fluorescent microscope with a fibre optic eyepiece and a simple photometric system. It was evaluated in direct immunofluorescence assays of Bacillus anthracis and Bacillus cereus spores immobilised on multispot microscope slides. From measurements of stable fluorescent crystals comparable in size to the spores, it was inferred that the fluorescence intensity of a stained bacterium could be measured with good precision. Fluctuation of a exciting light from a mercury vapour lamp did not contribute significantly to the distribution of fluorescence measurements obtained when samples of 20 spores were assessed. Attempts to correlate spore size with fluorescence intensity suggest that spore fluorescence does not increase in a 1 : 1 ratio with surface area; it is therefore possible that the density of antigenic sites on the surface decreases with increasing spore size. It is concluded that differences in the observed fluorescence of individual spores truly reflect differences in fluorescent antibody binding, but the relative contribution of antigenic variability and of artefacts of the staining procedure remain unknown.
Assuntos
Bacillus anthracis/imunologia , Bacillus cereus/imunologia , Citometria de Fluxo/métodos , Imunofluorescência , Animais , Anticorpos Antibacterianos , Antígenos de Bactérias , Tecnologia de Fibra Óptica , Coelhos , Esporos Bacterianos/imunologiaRESUMO
Quantitative indirect immunofluorescence assays for B. anthracis and B. cereus spores fixed on multispot microscope slides have been performed using a microfluorometer to measure the fluorescence of individual bacteria. A study has been made of variations of the indirect assay sequence, in which the washing operation between application of 1st and 2nd antibody types was omitted. In one modification the addition of the indirect antibody was deferred, and in the second the direct and indirect reagents were added simultaneously to the spores at the start of the incubation period. This simultaneous addition method shows promise for wide application in diagnostic serology. Evidence is presented that fluorescein-protein A (F-PA) can be successfully substituted for fluorescein/sheep anti-rabbit antibody (F-SAR) in the indirect spore assay, and that it is far more active than the F-SAR on a weight basis. About twice as many F-PA molecules as F-SAR molecules are bound to the spore at saturation.
Assuntos
Bacillus cereus/imunologia , Fluoresceínas/farmacologia , Imunofluorescência , Proteína Estafilocócica A/farmacologia , Animais , Anticorpos Antibacterianos , Bacillus anthracis/imunologia , Sítios de Ligação de Anticorpos , Cinética , Coelhos , Ovinos , Esporos/imunologia , Coloração e Rotulagem/métodosRESUMO
Indirect immunoradiometric assays (IRMA) for Bacillus anthracis spores are described in which the spores were heat fixed either on multispot microscope slides or on polyvinyl or polystyrene microtitre plates. Assays on plastic plates sometimes suffered from poor intra-experiment reproducibility. Signals were higher in assays in flat-bottomed microtitre wells than in assays in slides, but assay noise, due to non-specific absorption of antibody, was higher also, giving an overall disadvantage in signal-to-noise ratio. Elution of antibody with acid gave up to a doubling in signal-to-noise ratio. When small volumes of antibody reagents were used in assays in round-bottomed plastic wells, signal and noise characteristics were similar to those of the slide assay. While the assays on plastic wells tended to be less sensitive in terms of anti-spore antibody concentration or the number of spores detectable, the larger volume of antigen suspension that could be used in plastic wells gave them an advantage in terms of the minimum antigen concentration detectable.
Assuntos
Anticorpos Antibacterianos/análise , Radioimunoensaio/métodos , Antígenos de Bactérias/análise , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Bacillus anthracis/fisiologia , Imunofluorescência , Radioimunoensaio/instrumentação , Esporos Bacterianos/imunologiaRESUMO
Flow immunofluorescence (FIF) techniques were established for the specific detection of the bacteria Escherichia coli, Legionella pneumophila and Bacillus anthracis spores after staining with fluorescein-conjugated antibacterial antibody. For each bacterial type, a comparison was made of gating on narrow forward angle (NFA) light scatter and on the red fluorescence (Red Flu) signal available from staining with the nucleic acid dye propidium iodide. No universal gating method was found, since Bacillus spores did not take up propidium iodide and only a part of the Legionella population gave detectable NFA scatter signals. The efficiency of detecting bacteria stained with antibody remained constant with differing concentrations of the specific bacterium, and the estimate of the count for specific bacteria expressed as a fraction of the total cytometer count fell sharply with bacterial concentration. This effect was apparently due to cytometer noise inherent in the high sensitivity of detection needed for particles as small as these bacteria. The noise did not originate in the photomultipliers and was evidently the result either of light scatter from sub-micron particles in the sheath fluid or scatter from optical components. Part of the noise could be removed by selective gating, but there remained a noise component overlapping with the NFA scatter and Red Flu signals from the heterologous bacteria, i.e., those not stained with specific antibody. In consequence, at the low bacterial concentrations used no meaningful cytometer count could be obtained for the excess of the unstained bacteria and the proportion of specific bacteria in the mixed population could not, therefore, be calculated.
Assuntos
Anticorpos Antibacterianos/análise , Técnicas Bacteriológicas , Citometria de Fluxo/métodos , Imunofluorescência , Bacillus anthracis/análise , Escherichia coli/análise , Legionella/análise , Luz , Microscopia de Fluorescência/métodos , Propídio , Espalhamento de Radiação , Esporos Bacterianos/análise , Coloração e RotulagemRESUMO
Bacillus anthracis spores and Escherichia coli were stained with fluorescein-conjugated antibody using direct and indirect methods, then analyzed by means of a commercial flow cytometer. To reduce the cytometer's fluorescence component resulting from unreacted conjugate, reaction mixtures were either diluted or were centrifuged through a sucrose solution using a moving zone technique. Evidence is produced that the fluorescence statistics for centrifuged samples closely represent the fluorescence distribution of stained single bacteria in the reaction mixture at the end of incubation; in particular, centrifugation did not cause aggregation of bacteria. Centrifugation is proposed as more effective than mere dilution for use with a wide range of bacterial concentrations, and the moving zone technique is to be preferred to conventional centrifugation in which bacteria tend to aggregate in the pellet. In indirect assays, it was shown that the washing step after reaction with antibacterial antibody may be omitted. The performance of direct and indirect staining methods was compared, including the use of either Staphylococcus aureus protein A or polyclonal sheep anti-rabbit antibody as the indirect reagent. When the bacterial concentration in reaction mixtures was increased the median fluorescence intensity fell, indicating that specific antibody had become limiting at low concentrations of the polyclonal antibody preparations. The implications of this for the design of flow cytometry assays of bacteria are discussed.
Assuntos
Bactérias/isolamento & purificação , Citometria de Fluxo , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/análise , Bacillus anthracis/imunologia , Centrifugação , Escherichia coli/imunologia , ImunofluorescênciaRESUMO
Six methods for the purification of immunoglobulin G (IgG) from serum were compared, using rabbit antiserum to Bacillus anthracis spores as a model. Antibody activity was monitored by a solid-phase immunoradiometric assay (IRMA). Salt precipitation/ion exchange chromatography and ethanol precipitation both resulted in IgG of high purity but there was considerable inactivation of antibody. Salt precipitation/affinity chromatography gave poor yields of antibody. PEG precipitation and gel filtration of Sephacryl S-300 gave moderate yields and purity of IgG, with little evidence of antibody inactivation. Salt precipitation was marginally more destructive than the last 2 methods, but is recommended for routine use on grounds of its simplicity. Should IgG prepared by salt precipitation prove inadequate for particular applications, gel filtration is recommended since it allows the balance of yield and purity to be altered at will.
Assuntos
Imunoglobulina G/isolamento & purificação , Animais , Bacillus anthracis/imunologia , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Eletroforese em Acetato de Celulose , Imunoglobulina G/imunologia , Coelhos/imunologia , Radioimunoensaio/métodos , Esporos Bacterianos/imunologiaRESUMO
Preimplantation mouse embryos, cultured in vitro and those freshly flushed from the reproductive tract, exhibit a switch in energy substrate preference, from pyruvate during the early preimplantation stages, to glucose at the blastocyst stage. Although the biochemical basis of this phenomenon is quite well characterized, its timing and possible association with developmental factors have not been considered. We have therefore examined the role of five developmental factors in determining the timing of the switch, namely: (1) embryo age (in hours post hCG); (2) developmental stage; (3) cytokinesis; (4) cell number; and (5) activation of the embryonic genome. One-cell embryos, which develop more slowly than 2-cell embryos in vitro, were used to investigate the role of embryo age and developmental stage. Cytochalasin D, which inhibits cytokinesis and delays the timing of compaction and cavitation, was used to investigate the role of cell division and developmental stage. Finally, transcription of the embryonic genome was examined with the inhibitor, alpha-amanitin. Pyruvate and glucose consumption by single embryos were measured using a noninvasive ultramicrofluorometric technique. The results showed that the timing of the switch in energy substrate preference is precisely regulated in the mouse preimplantation embryo. Activation of the embryonic genome is a prerequisite for the switch and its timing is closely associated with developmental stage, specifically compaction and/or cavitation. Cell number, cytokinesis and embryo age appeared to be unrelated to the timing of the switch. These conclusions may well be extrapolated to other species, since an increase in net glucose uptake, if not always at the expense of pyruvate, is a feature of preimplantation embryo metabolism in all mammals studied.