Remote-refocusing light-sheet fluorescence microscopy enables 3D imaging of electromechanical coupling of hiPSC-derived and adult cardiomyocytes in co-culture.

Improving cardiac function through stem-cell regenerative therapy requires functional and structural integration of the transplanted cells with the host tissue. Visualizing the electromechanical interaction between native and graft cells necessitates 3D imaging with high spatio-temporal resolution and low photo-toxicity. A custom light-sheet fluorescence microscope was used for volumetric imaging of calcium dynamics in co-cultures of adult rat left ventricle cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes. Aberration-free remote refocus of the detection plane synchronously to the scanning of the light sheet along the detection axis enabled fast dual-channel 3D imaging at subcellular resolution without mechanical sample disturbance at up to 8 Hz over a ∼300 µm × 40 µm × 50 µm volume. The two cell types were found to undergo electrically stimulated and spontaneous synchronized calcium transients and contraction. Electromechanical coupling improved with co-culture duration, with 50% of adult-CM coupled after 24 h of co-culture, compared to 19% after 4 h (p = 0.0305). Immobilization with para-nitroblebbistatin did not prevent calcium transient synchronization, with 35% and 36% adult-CM coupled in control and treated samples respectively (p = 0.91), indicating that electrical coupling can be maintained independently of mechanotransduction.

Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging.

We report the characterisation of gated optical image intensifiers for fluorescence lifetime imaging, evaluating the performance of several different prototypes that culminate in a new design that provides improved spatial resolution conferred by the addition of a magnetic field to reduce the lateral spread of photoelectrons on their path between the photocathode and microchannel plate, and higher signal to noise ratio conferred by longer time gates. We also present a methodology to compare these systems and their capabilities, including the quantitative readouts of Förster resonant energy transfer.

Arthroscopic reattachment of osteochondritis dissecans cartilage flaps of the femoropatellar joint: long-term results.

REASONS FOR PERFORMING STUDY: Long-term efficacy of arthroscopic cartilage reattachment for the treatment of osteochondritis dissecans (OCD) lesions in the equine femoropatellar joint is unknown. OBJECTIVE: To evaluate radiographic outcome and long-term performance of horses undergoing OCD reattachment. HYPOTHESIS: Separated OCD cartilage flaps may be reincorporated into the joint surface by reattachment rather than flap removal. METHODS: Polydioxanone pins were utilised arthroscopically to reattach OCD lesions in 40 of 44 joints from 27 horses. Cartilage was reattached when it had persisting perimeter continuity, the surface was not deeply fissured or irregular, and the cartilage was not protuberant or extensively mineralised. Bone marrow aspirate concentrate was grafted to additional areas denuded of cartilage or alongside reattached cartilage. RESULTS: Breeds included Thoroughbred (n = 18), Quarter Horse (n = 4), Warmblood (n = 3), Standardbred (n = 1) and Arabian (n = 1). Mean age was 9.7 months. Radiographic lesion length was 1.5-6.3 cm. Reattachment alone was used in 32 of 44 affected joints, a combination of debridement and reattachment in 8 joints and debridement alone in 4 joints. One horse was destroyed due to tendon laceration. Of the remaining 26 horses, mean duration of follow-up was 15.6 months (range 2 months-12 years). Radiographic resolution of OCD lesions treated with reattachment was significantly improved at 6 months. Twenty horses had long-term performance data, of which 19 were sound and had reached intended athletic potential. One horse remained lame, and an additional 6 were sound but remained unbroken or were convalescing. Thus, an overall success rate based upon continued soundness in performing horses was 95% (19/20). CONCLUSION: Cartilage flap reattachment can salvage OCD cartilage by integration with the underlying bone. POTENTIAL RELEVANCE: Extensive OCD cartilage flaps may be salvaged by reattachment which can result in normal radiographic subchondral bone contour and long-term athletic performance.

Arthroscopic treatment of meniscal cysts in the horse.

REASONS FOR PERFORMING STUDY: To describe the clinical symptoms, treatment, and outcome of meniscal cysts in horses. These structures have not been previously described in the literature as a potential cause of lameness in the horse. HYPOTHESIS: Meniscal cysts are an uncommon condition of the femorotibial joint but can be a significant cause of lameness. Symptoms can be resolved by arthroscopic excision. METHODS: Records of horses diagnosed with meniscal cysts and treated by cyst excision and meniscal debridement at 2 surgical practices were reviewed. Clinical outcome was determined by repeat veterinary examination and contact with owner. RESULTS: Seven cases of meniscal cyst were treated with arthroscopic cyst excision and meniscal debridement. Five of 7 horses had lameness attributable to femorotibial joint pathology, while the remaining 2 horses had meniscal cysts found incidentally during diagnostic arthroscopy for the treatment of osteochondritis dissecans of the lateral trochlear ridge of the femur. Five of 6 horses with long-term follow-up were sound and a 7th horse was improved 11 months after surgery. CONCLUSIONS AND POTENTIAL RELEVANCE: Meniscal cysts, while uncommon, can be associated with progressive lameness in the horse. Surgical excision of the cysts results in resolution or improvement of symptoms, without evidence of recurrence on follow-up examination.

FK506 promotes adenosine release from endothelial cells via inhibition of adenosine kinase.

The immunosuppressants, cyclosporin A and tacrolimus (FK506) induce an increase in plasma levels of adenosine and mimic ischemic preconditioning. However, the mechanism of action of the two drugs on adenosine metabolism is not clear. Since inhibition of adenosine kinase promotes an increase in endogenous adenosine release, we tested a hypothesis that FK506 induces adenosine release via inhibition of adenosine kinase activity. In cultured endothelial cells, FK506 enhanced release of tracer adenosine and inhibited uptake of tracer adenosine. It also reduced adenosine kinase activity of the cell membrane fraction. In addition, FK506 does not inhibit membrane transport of tracer adenosine. These observations indicate that FK506 inhibits in situ adenosine kinase activity in endothelial cells. Other cell signaling inhibitors were found to inhibit adenosine uptake via inhibition of adenosine transport. In conclusion, FK506 promotes adenosine release from endothelial cells by a novel mechanism involving inhibition of adenosine kinase activity associated with the membrane.

Cellular and molecular actions of adrenomedullin in glomerular mesangial cells.

Adrenomedullin (AM), a potent vasodilatory and hypotensive peptide produces several biological outcomes in glomerular mesangial cells. Mesangial cells are important in the pathogenesis of glomerulonephritis, and therefore the actions of AM on mesangial cells have important clinical and therapeutic implications. This minireview describes the various actions of AM on mesangial cell function and the signal transduction mechanisms involved. As in other systems, most actions of AM can be explained by increase in cAMP levels in the cell, although a few exceptions remain. The fact that most data obtained to date has been in culture, the physiological significance of the actions of AM in mesangial cells is discussed.

A holistic approach for protein secondary structure estimation from infrared spectra in H(2)O solutions.

We present an improved technique for estimating protein secondary structure content from amide I and amide III band infrared spectra. This technique combines the superposition of reference spectra of pure secondary structure elements with simultaneous aromatic side chain, water vapor, and solvent background subtraction. Previous attempts to generate structural reference spectra from a basis set of reference protein spectra have had limited success because of inaccuracies arising from sequential background subtractions and spectral normalization, arbitrary spectral band truncation, and attempted resolution of spectroscopically degenerate structure classes. We eliminated these inaccuracies by defining a single mathematical function for protein spectra, permitting all subtractions, normalizations, and amide band deconvolution steps to be performed simultaneously using a single optimization algorithm. This approach circumvents many of the problems associated with the sequential nature of previous methods, especially with regard to removing the subjectivity involved in each processing step. A key element of this technique was the calculation of reference spectra for ordered helix, unordered helix, sheet, turns, and unordered structures from a basis set of spectra of well-characterized proteins. Structural reference spectra were generated in the amide I and amide III bands, both of which have been shown to be sensitive to protein secondary structure content. We accurately account for overlaps between amide and nonamide regions and allow different structure types to have different extinction coefficients. The agreement between our structure estimates, for proteins both inside and outside the basis set, and the corresponding determinations from X-ray crystallography is good.

Microalbuminuria in urban Zimbabwean women.

The prevalence of microalbuminuria (MAU) in African populations has not been reported, nor has the relationship between MAU and hypertension been reported for these populations. We collected spot urine samples from 370 women, 25 years and older as a part of a population-based, cross-sectional blood pressure survey in an urban community in Zimbabwe and analysed the samples for albumin and beta2-microglobulin. The age-adjusted prevalence of hypertension was 30% for women 25 years and older in this community. After excluding the samples with hematuria (11%), the prevalence of MAU (3.0 < or = albumin-to-creatinine ratio (ACR, mg/mmol) <25.0) in the study population was 9%. When age-adjusted to the population in the community, the prevalence was 8% among women 25 years and older. The prevalence of MAU was substantially higher in hypertensive (HT) than in normotensive (NT) women (16% vs 4%, P<0.001). A significantly higher level of log ACR in HT was found in each age group except the youngest age group (age 25-34). In age-adjusted multiple regression, percent fat mass was negatively associated with log ACR (beta = -1. 18, 95% CI (-0.23, -2.21), P = 0.02). In a similar regression analysis, higher log beta8-microglobulin-to-creatinine ratio was very strongly associated with higher log ACR (beta = 0.34, 95% CI (0.25, 0.43), P<0.0001) and significantly associated with lower percent fat mass (beta = -1.02, 95% CI (-0.25, -1.8), P = 0.01). These results suggest that MAU is frequently caused by hypertension, but that other diseases may contribute to its presence.

Economic development and women's blood pressure: field evidence from rural Mashonaland, Zimbabwe.

A survey of 515 non-pregnant women at 12 geographically chosen research sites in rural Mashonaland shows significant differences in mean blood pressure, controlled by age cohorts. Three levels of economic development are identified: (1) the traditional economy on communal lands, with lowest blood pressure, (2) the wage economy in areas of large-scale commercial agriculture, with elevated blood pressure and (3) the wage economy in mining areas, with the highest elevation of blood pressure. The area is dominated by the primate city, Harare, up to distances of 300 km and beyond, from which forces of change and modernization emanate. It is seen that potassium, sodium and the sodium potassium ratio, are distance-related to Harare and that women's blood pressures tend to follow suit. The rise of body sodium in young persons at risk, often accompanied by declining potassium intake and other changes of modernization, suggest that more attention should be focused on rural areas in Africa, now in the throes of economic change.

Correlates of blood pressure in an urban Zimbabwean population and comparison to other populations of African origin.

We have evaluated the relationship between systolic blood pressure (SBP) and age, body mass index (BMI), waist circumference, sodium to potassium ratio (Na/K), and tobacco use in an urban African population. We conducted a random, population-based, cross-sectional survey of people 25 years and older in Marondera, Zimbabwe, with over-sampling in older age groups (n = 775), using a method comparable to that used in International Collaborative Study on Hypertension in Blacks (ICSHIB). The age-adjusted prevalences of hypertension in Marondera (SBP >/=140/DBP >/=90/antihypertensive medication) were 30% for women and 21% for men. The average BMI was 26.3 kg/m2 for women and 21.4 kg/m2 for men. The prevalence of hypertension had a steep association with age and in women ranged from 15% (25-34 years) to 63% (55 years and over) and in men from 9% to 47%. No tobacco use in women and greater Na/K ratio in spot urines in men were significantly associated with an increased SBP. In both men and women the levels of hypertension and SBP were strongly positively associated with BMI, although the relationship appeared to plateau in women with a BMI greater than >/=25 kg/m2. At a given BMI, men and women had similar SBPs and prevalences of hypertension. There is a very high prevalence of hypertension among urban Zimbabweans, particularly among women. Under the assumption the studies are comparable, the prevalence of hypertension in Zimbabwean women (41%) and men (26%) after age adjustment to the ICSHIB populations, appeared higher than almost all of the ICSHIB populations, including those with higher average body mass indexes. Journal of Human Hypertension (2000) 14, 65-73.

Learning the regulation of peripheral blood flow.

Students can learn a great deal about the peripheral circulation when teaching is based on five building blocks: hemodynamic principles, neurohumoral control, and three elements of local control of blood flow (metabolic, myogenic, and paracrine). Study of a particular special circulation starts with the application of these building blocks in the context of the function of that tissue. For example, control of skin blood flow is largely concerned with regulation of body temperature (neurohumoral control) and the response to injury (paracrine control). Regulation of coronary blood flow is almost entirely a matter of meeting the metabolic needs of the myocardium (metabolic control). By mixing and matching the five building blocks and keeping in mind the special functions of a particular tissue, students can master the peripheral circulation efficiently.

Effect of variation in environmental temperature on blood pressure: is it important?

OBJECTIVES: To study the effect of variation in environmental temperature on blood pressure and pulse. DESIGN: Repeated blood pressure and pulse measurements in volunteers on two days when environmental temperatures differed by 10 degrees C. SETTING: Part of an ongoing community based study. SUBJECTS: 25 Black subjects (23 males and two females) volunteered from a population cohort participating in an ongoing longitudinal study examining cardiovascular risk factors in an urban African Black environment. MAIN OUTCOME MEASURE: Mean systolic and diastolic blood pressures and pulse rates. RESULTS: The systolic and diastolic blood pressures were significantly higher when recorded at 15 degrees C than at 25 degrees C, mean difference 32.2 +/- 4.2, p < 0.001 and 19.5 +/- 3.0 p < 0.001) for systolic and diastolic blood pressures respectively. The pulse rate per minute at 15 degrees C was significantly lower than at 25 degrees C (mean difference 11.1 +/- 3.2 p = 0.002). CONCLUSIONS: A decrease in environmental temperature by 10 degrees C appears to increase blood pressure. Awareness of this phenomenon is important, especially when surprisingly high blood pressures are observed during low ambient temperatures, to avoid over diagnosis of hypertension. This phenomenon, together with that already established of the white coat hypertension, may lead to the erroneous diagnosis of hypertension.

Long-term and high-temperature storage of supercritically-processed microparticulate protein powders.

PURPOSE: The long-term and high-temperature storage of dry, micron-sized particles of lysozyme, trypsin, and insulin was investigated. Subsequent to using supercritical carbon dioxide as an antisolvent to induce their precipitation from a dimethylsulfoxide solution, protein microparticles were stored in sealed containers at -25, -15, 0, 3, 20, 22, and 60 degrees C. The purpose of this study was to investigate the suitability of supercritical antisolvent precipitation as a finishing step in protein processing. METHODS: Karl Fisher titrations were used to determine the residual moisture content of commercial and supercritically-processed protein powders. The secondary structure of the dry protein particles was determined periodically during storage using Raman spectroscopy. The proteins were also redissolved periodically in aqueous buffers and assayed spectrophotometrically for biological activity and by circular dichroism for structural conformation in solution. RESULTS: Amide I band Raman spectra indicate that the secondary structure of the protein particles, while perturbed from that of the solution state, remained constant in time, regardless of the storage temperature. The recoverable biological activity upon reconstitution for the supercritically-processed lysozyme and trypsin microparticles was also preserved and found to be independent of storage temperature. Far UV circular dichroism spectra support the bioactivity assays and further suggest that adverse structural changes, with potential to hinder renaturation upon redissolution, do not take place during storage. CONCLUSIONS: The present study suggests that protein precipitation using supercritical fluids may yield particles suitable for long-term storage at ambient conditions.

Inorganic phosphate as regulator of adenosine formation in isolated guinea pig hearts.

This study evaluated cytosolic P(i) as an independent regulator of cardiac adenosine formation by dissociating changes in P(i) from changes in AMP and ADP. Myocardial high-energy phosphates (HEP), measured by (31)P nuclear magnetic resonance spectroscopy, were depleted acutely by perfusing isolated guinea pig hearts with 2-deoxyglucose (2-DG), and the effects of 2-DG were compared with a norepinephrine infusion producing similar changes in HEP. 2-DG treatment resulted in lower adenosine release (R(ado)) (54 +/- 18 vs. 622 +/- 199 pmol x min(-1) x g(-1)) and P(i) concentration ([P(i)]) (0.5 +/- 0.1 vs. 6.0 +/- 0.9 mM) than norepinephrine despite similar AMP concentration ([AMP]). Chronic phosphocreatine depletion produced by beta-guanidinopropionic acid feeding also reduced R(ado) and P(i) during hypoxia. Replacement of perfusate glucose and pyruvate with acetate increased R(ado) (from 39 +/- 12 to 356 +/- 100 pmol x min(-1) x g(-1)) and [P(i)] (from 2.0 +/- 0.5 to 5.1 +/- 0.6 mM) with no change in cytosolic [AMP]. Adenosine kinase isolated from guinea pig hearts was inhibited by [P(i)] values seen during hypoxia or hypoperfusion. We conclude that cytosolic [P(i)] can be an important regulator of cardiac adenosine formation through inhibition of adenosine kinase.

Evaluation of an electronic blood pressure device for epidemiological studies.

An objective method for measuring blood pressure would enhance the usefulness of comparative survey research in hypertension. Previous studies have demonstrated that inexpensive, commercially available machines can achieve satisfactory levels of precision. We evaluated one such device in three field settings. Among 64 untreated hypertensive patients, electronic readings both in the clinic and at home were well correlated with 24 h ambulatory readings (correlation ranged from 0.6 to 0.8), consistently better than measurements obtained by human observers. Evaluation as part of a quality control exercise in an ongoing random population surveys from four countries demonstrated average correlations of 0.92 for systolic and 0.85 for diastolic between human and electronic readings. Subsequent use in survey research involving 4000 individuals in three African communities demonstrated consistency of the electronic devices. These cross-sectional surveys, in which measurements were made by non-professionals, provided qualitative estimates of rural-urban variation in blood pressure, demonstrating the practical utility of the technique. If verified by other evaluative studies, the new generation of semi-automatic electronci blood pressure devices could markedly enhance the comparability of hypertension prevalence studies.

Comparison of the Omron HEM-713C automated blood pressure monitor with a standard ausculatory method using a mercury manometer.

OBJECTIVES: To compare the Omron HEM-713C automated blood pressure machine with the standard ausculatory method using a mercury manometer. DESIGN: Blood pressures of randomly selected subjects were measured using both the Omron HEM-713C and the mercury manometer. SETTING: Dombotombo surburb in Marondera, Zimbabwe. SUBJECTS: One hundred and sixteen subjects 25 years and above (47 males and 69 females) randomly selected in Marondera. MAIN OUTCOME MEASURE: Systolic blood pressure and diastolic blood pressure. RESULTS: The Omron HEM-713C passed with a grade B for both systolic and diastolic blood pressures when using the British Hypertension Society protocol. It also passed both systolic and diastolic criteria for Association of the Advancement of Medical Instrumentation. CONCLUSION: The Omron HEM-713C compares well with the standard mercury manometer, we therefore recommend its use in both research and clinical applications which require blood pressure measurements.

Precipitation of proteins in supercritical carbon dioxide.

Supercritical CO2 was used as an antisolvent to form protein particles that exhibited minimal loss of activity upon reconstitution. Organic protein solutions were sprayed under a variety of operating conditions into the supercritical fluid, causing precipitation of dry, microparticulate (1-5 microns) protein powders. Three proteins were studied: trypsin, lysozyme, and insulin. Amide I band Raman spectra were used to estimate the alpha-helix and beta-sheet structural contents of native and precipitate powders of each protein. Analysis of the Raman spectral revealed minimal (lysozyme), intermediate (trypsin), and appreciable (insulin) changes in secondary structure with respect to the commercial starting materials. The perturbations in secondary structure suggest that the most significant event during supercritical fluid-induced precipitation involved the formation of beta-sheet structures with concomitant decreases of alpha-helix. Amide I band Raman and Fourier-transform infrared (FTIR) spectra indicate that higher operating temperatures and pressures lead to more extensive beta-sheet-mediated intermolecular interactions in the precipitates. Raman and FTIR spectra of redissolved precipitates are similar to those of aqueous commercial proteins, indicating that conformational changes were reversible upon reconstitution. These results suggest that protein precipitation in supercritical fluids can be used to form particles suitable for controlled release, direct aerosol delivery to the lungs, and long-term storage at ambient conditions.

Prevalence of hypertension among women in rural Zimbabwe: a comparison of pregnant and non-pregnant women.

OBJECTIVES: To determine the prevalence of pregnancy induced hypertension (PIH) in rural Zimbabwe. DESIGNS: A blood pressure survey conducted in three socio-economic strata of rural Zimbabwe, communal lands, commercial farms and mining areas. SETTING: Rural Zimbabwe, Mashonaland West Province. SUBJECTS: 627 pregnant (> 20 weeks gestation) and 483 non pregnant (ages 16 to 45) women. MAIN OUTCOME MEASURE: Prevalence of PIH. RESULTS: PIH was defined in two ways: 1. A combination of > or = 2+ proteinuria and systolic and/or diastolic pressure > or = SD above the mean for all pregnant women (> or = 123/75 mmHg). 2. > or = 1+ proteinuria and blood pressure of 140 and/or 90 mmHg. Eight women met definition 1 and seven met definition 2, giving a prevalence of 0.8pc; 1.3pc and 3.2pc in communal lands, commercial farms and mines respectively. In non-pregnant women the prevalence of hypertension (> or = 160 and/or 95 mmHg) was 3.5pc; 5.4pc and 15.1pc in communal lands, commercial farms and mines respectively. CONCLUSION: PIH is rare in rural Zimbabwe. Hypertension among non-pregnant women of child bearing age is most common in mining communities which have many similarities to urban environments.

Adenosine formation during hypoxia in isolated hearts: effect of adrenergic blockade.

Adrenergic receptor blockade has been reported to decrease cardiac adenosine formation and release during hypoxia. We wished to determine whether this occurs by an improvement in the energy supply/demand ratio. Isolated guinea pig hearts were perfused at a constant pressure of 50 mm Hg. Hypoxia (30% O2) was maintained for 20 min while adenosine release and venous PO2 were measured in the coronary venous effluent. beta-adrenergic blockade with 5 microM atenolol did not change hypoxic adenosine release (Control: 15.6 +/- 2.7, Atenolol: 23.6 +/- 5.7 nmol/g/20 min). Addition of 6 microM phentolamine with atenolol significantly reduced hypoxic adenosine release (4.4 +/- 1.4 nmol/g/20 min, P < 0.05). Atenolol was without hemodynamic effects, but addition of phentolamine reduced left ventricular pressure development, heart rate, and oxygen consumption prior to hypoxia. Atenolol plus phentolamine did not change venous PO2 during hypoxia. Treatment with phenoxybenzamine (1 microM) plus atenolol also reduced adenosine release (7.4 +/- 0.8 nmol/g/20 min). Control experiments and atenolol plus phentolamine experiments were repeated using 31P-NMR to measure high energy phosphates. Adrenergic blockade had no effect on phosphate concentrations during normoxia, but resulted in higher [PCr], lower [P(i)] and higher phosphorylation potentials during hypoxia. Adrenergic blockade also prevented the hypoxia-induced rise in intracellular [H+], [AMP] and [ADP] seen in control hearts. The changes in phosphorylation potential are correlated with similar changes in adenosine release in adrenergically intact hearts. We conclude that the primary effect of adrenergic blockade during hypoxia is a reduction in ATP use due to alpha-receptor blockade. This leads to improved high energy phosphate concentrations during hypoxia and a reduction in adenosine formation.