Measurement of the fluorescence lifetime of GFP in high refractive index levitated droplets using FLIM.

Green fluorescent protein (GFP) is a widely used fluorescent probe in the life sciences and biosciences due to its high quantum yield and extinction coefficient, and its ability to bind to biological systems of interest. This study measures the fluorescence lifetime of GFP in sucrose/water solutions of known molarity in order to determine the refractive index dependent lifetime of GFP. A range of refractive indices from 1.43-1.53 were probed by levitating micron sized droplets composed of water/sucrose/GFP in an optical trap under well-constrained conditions of relative humidity. This setup allows for the first reported measurements of the fluorescence lifetime of GFP at refractive indices greater than 1.46. The results obtained at refractive indices less than 1.46 show good agreement with previous studies. Further experiments that trapped droplets of deionised water containing GFP allowed the hygroscopic properties of GFP to be measured. GFP is found to be mildly hygroscopic by mass, but the high ratio of molecular masses of GFP to water (ca. 1500 : 1) signifies that water uptake is large on a per-mole basis. Hygroscopic properties are verified using brightfield microscope imaging, of GFP droplets at low and high relative humidity, by measuring the humidity dependent droplet size. In addition, this experiment allowed the refractive index of pure GFP to be estimated for the first time (1.72 ± 0.07). This work provides reference data for future experiments involving GFP, especially for those conducted in high refractive index media. The work also demonstrates that GFP can be used as a probe for aerosol studies, which require determination of the refractive index of the aerosol of any shape.

Fluorescence lifetime imaging of optically levitated aerosol: a technique to quantitatively map the viscosity of suspended aerosol particles.

We describe a technique to measure the viscosity of stably levitated single micron-sized aerosol particles. Particle levitation allows the aerosol phase to be probed in the absence of potentially artefact-causing surfaces. To achieve this feat, we combined two laser based techniques: optical trapping for aerosol particle levitation, using a counter-propagating laser beam configuration, and fluorescent lifetime imaging microscopy (FLIM) of molecular rotors for the measurement of viscosity within the particle. Unlike other techniques used to measure aerosol particle viscosity, this allows for the non-destructive probing of viscosity of aerosol particles without interference from surfaces. The well-described viscosity of sucrose aerosol, under a range of relative humidity conditions, is used to validate the technique. Furthermore we investigate a pharmaceutically-relevant mixture of sodium chloride and salbutamol sulphate under humidities representative of in vivo drug inhalation. Finally, we provide a methodology for incorporating molecular rotors into already levitated particles, thereby making the FLIM/optical trapping technique applicable to real world aerosol systems, such as atmospheric aerosols and those generated by pharmaceutical inhalers.

Cloud condensation nucleation activities of calcium carbonate and its atmospheric ageing products.

Aerosol particles can serve as cloud condensation nuclei (CCN) to form cloud droplets, and its composition is a main factor governing whether an aerosol particle is an effective CCN. Pure mineral dust particles are poor CCN; however, changes in chemical composition of mineral dust aerosol particles, due to heterogeneous reactions with reactive trace gases in the troposphere, can modify their CCN properties. In this study we investigated the CCN activities of CaCO3 (as a surrogate for mineral dust) and its six atmospheric ageing products: Ca(NO3)2, CaCl2, CaSO4, Ca(CH3SO3)2, Ca(HCOO)2, and Ca(CH3COO)2. CaCO3 has a very low CCN activity with a hygroscopicity parameter (κ) of 0.001-0.003. The CCN activities of its potential atmospheric ageing products are significantly higher. For example, we determined that Ca(NO3)2, CaCl2 and Ca(HCOO)2 have κ values of ∼0.50, similar to that of (NH4)2SO4. Ca(CH3COO)2 has slightly lower CCN activity with a κ value of ∼0.40, and the κ value of CaSO4 is around 0.02. We further show that exposure of CaCO3 particles to N2O5 at 0% relative humidity (RH) significantly enhances their CCN activity, with κ values increasing to around 0.02-0.04. Within the experimental uncertainties, it appears that the variation in exposure to N2O5 from ∼550 to 15,000 ppbv s does not change the CCN activities of aged CaCO3 particles. This observation indicates that the CaCO3 surface may be already saturated at the shortest exposure. We also discussed the atmospheric implications of our study, and suggested that the rate of change in CCN activities of mineral dust particles in the troposphere is important to determine their roles in cloud formation.

Pathophysiologic studies in human rabies.

Six patients with proved rabies were studied with a combination of clinical, physiologic and pathologic technics. Three were given a type of intensive care but died with evidence of respiratory failure. Although circulatory failure did not develop in any of the six patients, three had supraventricular arrhythmias: interstitial myocarditis was found in one of these and rabies virus was isolated from the myocardium of another. Inspiratory muscle spasm was the dominant clinical feature in all cases. This occurred as part of the hydrophobic response and followed stimulation of the upper respiratory tract and skin. Hydrophobia may represent an exaggerated respiratory tract irritant reflex with associated arousal. Later in the course of the disease, various patterns of periodic and ataxic breathing were observed. Widespread brain stem encephalitis was discovered at autopsy, with particular involvement of the neighborhood of the nucleus ambiguous in two of three patients examined. In one patient cerebral metabolism was grossly abnormal, with greatly reduced cerebral oxygen consumption suggesting irreversible brain damage. Respiratory and circulatory disturbances may well be immediate causes of death in patients with rabies, but the present studies reemphasize the severity of the encephalitis which remains the ultimate barrier to survival. In the developing countries in which rabies is still a major problem and in which the cost precludes intensive care, the clinical management of rabies can aim only to reduce suffering by heavy sedation.

Decline of Haemophilus influenzae type b disease in a region of high risk: impact of passive and active immunization.

Haemophilus influenzae type b (Hib) is a major cause of serious childhood bacterial infections. Before 1989 Alaska Native infants in the Yukon Kuskokwim Delta (YKD) had the highest recorded Hib disease rate, 2960:100,000 in children less than 1 year of age with 6 to 35 (mean, 13) cases/year between 1980 and 1988. In July, 1989, Alaska Area Native Health Service initiated a passive immunization project in the YKD using bacterial polysaccharide immunoglobulin (BPIG) administered at 3-month intervals to prevent Hib infections in infants less than 13 months of age. On January 1, 1991, after licensure of Hib conjugate vaccines for infants, the program was modified to a passive-active strategy using BPIG at birth and PedvaxHIB at 2, 4 and 12 months of age. Between July 1, 1989, and December 31, 1990, 80% of YKD children less than 1 year of age received at least 1 dose of BPIG. During this period there were 7 Hib cases in this age group, but only 1 of the cases had received any BPIG. Between January 1, 1991, and December 31, 1992, 4 Hib cases occurred in 2 YKD children. During the combined period, July 1, 1989, to December 31, 1992, the incidence of Hib disease for infants less than 1 year of age was 302:100,000. A dramatic decrease in Hib disease was observed in this high incidence region concurrent with implementation of passive and passive-active immunization strategies.

Bites by the night adder (Causus maculatus) and burrowing vipers (genus Atractaspis) in Nigeria.

Nineteen patients proven to have been bitten by the small African adders Causus maculatus, Atractapis dahomeyensis and A. microlepidota were studied in the Nigerian savanna region. One of the patients bitten by C. maculatus was drowsy, hypotensive and flaccid on admission but recovered without treatment. Mild or moderate local swelling, local lymphadenitis and mild fever were the only other features in this group. None of the patients bitten by Atractaspis had signs of systemic envenoming apart from moderate fever. Local blistering appeared in two cases but did not progress to necrosis. No patient showed any disturbance of blood coagulation, or evidence of spontaneous hemorrhage or of cranial nerve lesions. The small literature on the effects of Causus and Atractaspis venoms in man and in laboratory animals is reviewed. It appears that bites by these species are very unlikely to cause serious ill effects. A few deaths from Atractaspis bites have been reported, but the danger from these species has been exaggerated.

Kikuchi-Fujimoto disease: a report of two cases and an overview.

Kikuchi-Fujimoto disease (KFD) has been widely reported from Japan and sporadically from many parts of the world including Saudi Arabia, since its original description in 1972 but the disease remains poorly known by clinicians. In this paper we report two Saudi patients seen in Tabuk, Saudi Arabia. One was a 36-year-old Saudi man and the other a 16-year-old Saudi girl. Both presented with cervical lymphadenopathy and pyrexia. Histological examination of biopsy material from both showed classical features of KFD. Other laboratory findings were unremarkable except for leucopenia. Following excision biopsy both patients recovered without sequelae. KFD is a self-limiting process of uncertain aetiology that predominantly affects young women aged 20-30 years. We review the pathology, clinical featuers and possible aetiology of this interesting disease, which may well be underdiagnosed. Increased awareness of KFD will minimize the risk of confusing this entity with malignant lymphoma or other serious conditions.

RSV-associated hospitalizations in Alaska Native infants.

PURPOSE: Retrospective reviews for 1986-1992 suggested that Alaska Native children experience high rates of respiratory syncytial virus (RSV)-associated hospitalization; however, the epidemiology of RSV infections has been poorly characterized. METHODS: A prospective hospital-based surveillance study was undertaken to determine rates of RSV-associated hospitalization in Alaska Native children < 36 months from the Yukon-Kuskokwim Delta. RESULTS: During the first study year, October 1993 to September 1994, there were 40 RSV cases (hospitalization rate, 53/1,000 infants < 1 year of age); however, during the second year, October 1994 to September 1995, there were 251 RSV cases (hospitalization rate, 294/1,000 infants). An unusually high proportion, 12%, of RSV cases were < 1 month of age. Disease severity was higher for children with a history of prematurity, heart, or lung disease (p = .001, X2 analysis). Of 255 cell cultures during 1994-1995, 190 were RSV-positive, 11 were positive for influenza, 4 for adenovirus, and 1 for parainfluenza. This study demonstrates wide seasonal variation in a population with an extremely high RSV hospitalization rate; increased disease severity associated with young age and pre-existing medical conditions; and co-circulation of RSV with other viruses.

Possible hepatoxicity of tocainide.

A case of a woman on tocainide who developed abnormal liver function. A causal relationship is postulated.

Observations in man of hypoglycaemia during selective and non-selective beta-blockade.

The acute hypoglycaemic reaction is accompanied by a rise in systolic and a slight fall in diastolic blood pressure and a tachycardia. In contrast, during beta-blockade with propranolol there is a rise of both systolic and diastolic blood pressures and bradycardia. Restoration of blood glucose to normal is delayed. With metoprolol there is a lesser increase in diastolic blood pressure and a slight tachycardia. Restoration of the blood glucose to normal is little delayed. When patients liable to hypoglycaemia require a beta-blocking agent, it is suggested that a selective blocker such as metoprolol should be used.

Peri-partum cardiac failure.

The syndrome of peri-partum cardiac failure (PPCF) has been studied in 224 women seen in three years in Zaria, in northern Nigeria. A very high proportion were rural Hausa patients. There was a seasonal peak in July, and the incidence was about one per cent of deliveries. The risk increased with both age and parity. Symptoms began most commonly in the second week after delivery, and admission was commonest in the fourth. Typical signs of cardiac failure were found, and pulsus alternans, atrio-ventricular valvular incompetence, transient systemic hypertension and splenomegaly were common. The chest radiograph showed marked cardiomegaly, and extrasystoles and inverted T waves were often present in the electrocardiogram (ECG). Hypoalbuminaemia was common. Digoxin and diuretics were rapidly effective, causing a mean weight loss of 29 per cent in 15 days, resolution of hypertension, and a fall in the cardio-thoracic ratio (CTR) from 61 to 53 per cent. During the first year after diagnosis, the CTR became normal in 82 per cent of patients, and the ECG in 60 per cent. PPCF recurred, again with the same seasonal variation, after 19 per cent of subsequent pregnancies. During follow up for two to five years, 22 per cent of the women became hypertensive, and 11 per cent died. The prognosis was worst in those with an arrhythmia, hypertension, sustained cardiomegaly or aged 30 or more. Asymtomatic post-partum hypertension (PPHT) was found in 61 per cent of normal Hausa women, with a seasonal peak in May, especially in those with hypertension during pregnancy or labour, and twin pregnancies. Peri-partum cardiac failure may be due to the combined pressure load of PPHT, the volume load from eating the customary sodium-rich kanwa, and the cardiovascular demands of heat, both climatic and traditionally self-imposed.

Experience with multiple choice question papers in Nigeria. A practical guide to their use in a new medical school.

The application of MCQ examination techniques, in the context of a new medical school in tropical Africa, is described. The pitfalls are noted, the composition of papers discussed, and a simple method of marking is described. Some examples of questions are given, and their individual values are simply assessed. From our experience, there is no doubt that objective testing has a real place in a young medical school, where it can actually save time, as well as reducing variation in standards of examination.

Defaulters from follow-up after pre-partum cardiac failure.

The problem of default from follow-up prejudices the study of disease in Africa. During studies of 224 patients with pre-partum cardiac failure were found that 119 had defaulted from follow-up for six months or more, after an average period of 22 months from their admission. A sociologist tried to find all these patients at home and interviewed those who were found. If the patient was not found, information was sought from the village head and from friends and relatives. Thirteen patients had died. The 69 who were traced gave a total of 101 reasons for default. Domestic reasons prodominated, of which poverty, moving home and the difficulties of travel were the most important. Among personal reasons, feeling well was the commonest single reason, and was given by 19 patients. The results show that the problems of a subsistence agricultural economy dominate the reasons for default, and that such problem cannot be overcome without changes in society. Our findings are relevant in the care of all chronic diseases and throughout rural Africa.

Bites by puff-adder (Bitis arietans) in Nigeria, and value of antivenom.

Ten patients bitten by the puff-adder (Bitis arietans) were studied in the North of Nigeria. Six showed severe local signs, and four also had evidence of systemic envenoming, including spontaneous bleeding with thrombocytopenia, hypotension, and bradycardia. Two patients died after developing circulatory collapse and renal failure. Antivenom and intravenous fluid restored blood pressure in two hypotensive patients, and antivenom probably prevented the development of local necrosis in four others with massive local swelling. Victims of B arietans who have swelling of more than half the bitten limb or show signs of systemic envenoming should be given at least 80 ml of specific polyvalent antivenom and watched carefully for signs of circulatory collapse. Debridement of necrotic tissue may be necessary.

Perpartum cardiac failure. An explanation for the observed geographic distribution in Nigeria.

Peripartum cardiac failure (PPCF) is common in Zaria, in northern Nigeria, but has not been described elsewhere in Nigeria except in Ibadan. The geographic origin of a series of 224 patients with PPCF was studied in Zaria, and a survey of the syndrome as seen in hospitals and by physicians in the northern states of Nigeria was carried out; information was also gathered from medical and nursing students from various tribal groups in the same area. It was found that PPCF is only common in the areas of Hausa majority, mostly around Zaria and Malumfashi, where the postpartum practices of taking hot baths, lying on a hot bed, and taking large amounts of kanwa (a lake-salt rich in sodium) are pursued with great vigour. These customs may impose a critical load on a vulnerable myocardium, and it seems that tribe and tradition could well explain the high incidence of PPCF around Zaria.

Hormonal and substrate responses during recovery from hypoglycaemia in man during beta 1-selective and non-selective beta-adrenergic blockade.

Recovery from acute hypoglycaemia induced by the injection of insulin has been examined in six human subjects under control conditions, under non-selective beta blockade (propranolol) and under selective beta 1 blockade (metoprolol). The normal blood glucose recovery was biphasic with an initial rapid and a slower subsequent phase of recovery. The early recovery mechanism was unaffected by either form of beta blockade, but with propranolol the late phase of recovery was significantly prolonged. Rises in blood lactate and plasma free fatty acids following hypoglycaemia were markedly reduced by propranolol but to a much lesser degree with metoprolol. The counterregulatory hormonal responses of glucagon, cortisol and growth hormone were augmented appropriately for the prolonged hypoglycaemia associated with propranolol. Non-selective beta adrenergic blockade with propranolol is associated with an impairment of the late phase of blood glucose recovery from hypoglycaemia. The possible mechanisms of this impairment are discussed.

Bromocriptine suppression of the thyrotrophin response to thyrotrophin releasing hormone.

A single oral dose of 5 mg of bromocriptine significantly lowered the TSH response to 200 microgram TRH intravenously in eight healthy men compared with control experiments in the same subjects. This finding may be relevant in chronic bromocriptine therapy.

T, B and K cells in autoimmune thyroid disease.

The K-cell cytotoxic activity of peripheral blood lymphoid cells from 104 patients with autoimmune thyroid disease and from age and sex matched control subjects was measured using chicken erythrocytes as target cells. Patients with Hashimoto thyroiditis,primary hypothyroidism and thyrotoxicosis who were either newly diagnosed and untreated or had received therapy for less than or equal to 1 year showed a significant increase in K-cell cytotoxic activity. Patients who had received treatment for greater than 1 year and less than or equal to 5 years showed no such comparable increase in cytotoxic activity. Within the group of patients with untreated thyrotoxicosis it was found that K-cell cytotoxic activity was related to both goitre size and serum antibody titre. Thus patients with little or no goitre showed a highly significant elevation of cytotoxic activity whereas patients with moderate to large goitres gave values within the normal range. Similarly patients with no detectable serum thyroid autoantibodies showed high K-cell activity while patients with positive antibody titres did not. It was also shown that neither the absolute number nor the proportion of circulating T and B lymphocytes in patients with autoimmune thyroid disease as assessed by the sheep red cell rosette method and by indirect immunofluorescence was significantly different from that observed in the normal control population. No correlation was found between peripheral blood K-cell cytotoxic activity and the percentage of circulating null cells, i.e. 100-(percentage T + percentage B) in either patients or control subjects.