Direct writing of optical microresonators in a lab-on-a-chip for label-free biosensing.

Whispering gallery mode (WGM) resonators are promising optical structures for microfluidic label-free biosensors mainly due to their high sensitivity, but from a practical point of view they present numerous constraints that make their use in real laboratory diagnosis application difficult. Herein we report on a monolithic lab on a chip fabricated by a hybrid femtosecond laser micromachining approach, for label-free biosensing. It consists of a polymer WGM microresonator sensor integrated inside a glass microfluidic chip, presenting a refractive index change sensitivity of 61 nm per RIU. The biosensing capabilities of the device have been demonstrated by exploiting the biotin-streptavidin binding affinity, obtaining a measurable minimum surface density increase of 67 × 103 molecules per µm2.

Hydrodynamic synchronization of light driven microrotors.

Hydrodynamic synchronization is a fundamental physical phenomenon by which self-sustained oscillators communicate through perturbations in the surrounding fluid and converge to a stable synchronized state. This is an important factor for the emergence of regular and coordinated patterns in the motions of cilia and flagella. When dealing with biological systems, however, it is always hard to disentangle internal signaling mechanisms from external purely physical couplings. We have used the combination of two-photon polymerization and holographic optical trapping to build a mesoscale model composed of chiral propellers rotated by radiation pressure. The two microrotors can be synchronized by hydrodynamic interactions alone although the relative torques have to be finely tuned. Dealing with a micron sized system we treat synchronization as a stochastic phenomenon and show that the phase lag between the two microrotors is distributed according to a stationary Fokker-Planck equation for an overdamped particle over a tilted periodic potential. Synchronized states correspond to minima in this potential whose locations are shown to depend critically on the detailed geometry of the propellers.

Wave-guided optical waveguides.

This work primarily aims to fabricate and use two photon polymerization (2PP) microstructures capable of being optically manipulated into any arbitrary orientation. We have integrated optical waveguides into the structures and therefore have freestanding waveguides, which can be positioned anywhere in the sample at any orientation using optical traps. One of the key aspects to the work is the change in direction of the incident plane wave, and the marked increase in the numerical aperture demonstrated. Hence, the optically steered waveguide can tap from a relatively broader beam and then generate a more tightly confined light at its tip. The paper contains both simulation, related to the propagation of light through the waveguide, and experimental demonstrations using our BioPhotonics Workstation. In a broader context, this work shows that optically trapped microfabricated structures can potentially help bridge the diffraction barrier. This structure-mediated paradigm may be carried forward to open new possibilities for exploiting beams from far-field optics down to the subwavelength domain.

Examination of chromosome 7p22 candidate genes RBaK, PMS2 and GNA12 in familial hyperaldosteronism type II.

1. There are two types of familial hyperaldosteronism (FH): FH-I and FH-II. FH-I is caused by a hybrid CYP11B1/CYP11B2 gene mutation. The genetic cause of FH-II, which is more common, is unknown. Adrenal hyperplasia and adenomas are features. We previously reported linkage of FH-II to a approximately 5 Mb region on chromosome 7p22. We subsequently reported finding no causative mutations in the retinoblastoma-associated Kruppel-associated box gene (RBaK), a candidate at 7p22 involved in tumorigenesis and cell cycle control. 2. In the current study we investigated RBaK regulatory regions and two other candidate genes: postmeiotic segregation increased 2 (PMS2, involved in DNA mismatch repair and tumour predisposition) and guanine nucleotide-binding protein alpha-12 (GNA12, a transforming oncogene). 3. The GNA12 and PMS2 genes were examined in two affected (A1, A2) and two unaffected (U1, U2) subjects from a large 7p22-linked FH-II family (family 1). No mutations were found. 4. The RBaK and PMS2 distal promoters were sequenced to -2150 bp from the transcription start site for RBaK and-2800 bp for PMS2. Five unreported single nucleotide polymorphisms (SNPs) were found in subjects A1, A2 but not in U1 or U2; A(-2031 bp)T, T(-2030 bp)G, G(-834 bp)C, C(-821 bp)G in RBaK and A(-876 bp)G in PMS2. Additional affected and unaffected subjects from family 1 and from two other 7p22-linked FH-II families and 58 unrelated normotensive control subjects were genotyped for these SNPs. 5. The five novel SNPs were found to be present in a significant proportion of normotensive controls. The four RBaK promoter SNPs were found to be in linkage disequilibrium in the normal population. The RBaK promoter (-)2031T/2030G/834C/821T allele was found to be in linkage disequilibrium with the causative mutation in FH-II family 1, but not in families 2 and 3. The PMS2 promoter (-)876G allele was also found to be linked to affected phenotypes in family 1. 6. The RBaK and PMS2 promoter SNPs alter the binding sites for several transcription factors. Although present in the normal population, it is possible that the RBaK (-)2031T/2030G/834C/821T and PMS2 (-)876G alleles may have functional roles contributing to the FH-II phenotype in family 1.

Interfacial water structure controls protein conformation.

A phenomenological theory of salt-induced Hofmeister phenomena is presented, based on a relation between protein solubility in salt solutions and protein-water interfacial tension. As a generalization of previous treatments, it implies that both kosmotropic salting out and chaotropic salting in are manifested via salt-induced changes of the hydrophobic/hydrophilic properties of protein-water interfaces. The theory is applied to describe the salt-dependent free energy profiles of proteins as a function of their water-exposed surface area. On this basis, three classes of protein conformations have been distinguished, and their existence experimentally demonstrated using the examples of bacteriorhodopsin and myoglobin. The experimental results support the ability of the new formalism to account for the diverse manifestations of salt effects on protein conformation, dynamics, and stability, and to resolve the puzzle of chaotropes stabilizing certain proteins (and other anomalies). It is also shown that the relation between interfacial tension and protein structural stability is straightforwardly linked to protein conformational fluctuations, providing a keystone for the microscopic interpretation of Hofmeister effects. Implications of the results concerning the use of Hofmeister effects in the experimental study of protein function are discussed.

GI Epidemiology: nutritional epidemiology.

BACKGROUND: Nutritional epidemiology is the assessment of diet and its relationship to disease aetiology in populations. The choice of dietary assessment method depends on the disease pathology. Events such as cancer that are chronic and complicated by exposure time require methods that capture consumption patterns of populations over a period of years. Although several methods of dietary assessment exist for collecting information on groups of individuals, their application to epidemiologic studies requires an understanding of the effect of variability in nutrient intake, sources of measurement error, and statistical issues unique to the study of nutritional epidemiology. AIM: This review provides an overview of commonly-used methods of dietary assessments in epidemiologic studies, and identifies their strengths and limitations and application to epidemiologic study designs. It concludes with a brief discussion of assumptions of nutrient databases and objectives of energy-adjustment and measurement error correction models. CONCLUSIONS: Nutritional epidemiology has contributed significantly to our understanding of the relationships between diet and disease. Ongoing investigations that further characterize important exposure periods (early life, in utero) and clarify associations within the context of genetic susceptibility will continue to elucidate our understanding of the pathophysiology of complex diseases, and support future recommendations for disease prevention.

Dietary determinants of one-carbon metabolism and the risk of non-Hodgkin's lymphoma: NCI-SEER case-control study, 1998-2000.

The role of dietary one-carbon determinants remains largely unexplored for non-Hodgkin's lymphoma (NHL). In a population-based case-control study of non-African-American adult (aged 20-74 years) women and men from four US Surveillance, Epidemiology, and End Results study centers (Detroit, Michigan; Iowa; Los Angeles, California; and Seattle, Washington; 1998-2000), the authors examined folate; vitamins B2, B6, and B12; methionine; and a one-carbon antagonist, alcohol, in 425 incident NHL cases and 359 controls who completed a detailed food frequency questionnaire. Adjusted odds ratios and 95% confidence intervals were estimated by using unconditional logistic regression. Higher intake of one-carbon determinants from food was associated with a lower risk of NHL, but that for only vitamin B6 (highest vs. lowest quartile: odds ratio = 0.57, 95% confidence interval: 0.34, 0.95; p trend = 0.01) and methionine (odds ratio = 0.49, 95% confidence interval: 0.31, 0.76; p trend = 0.002) reached statistical significance. Folate from food was inversely associated with diffuse subtype (odds ratio = 0.47, 95% confidence interval: 0.23, 0.94; p trend = 0.03). The authors found no association between total (food plus supplement) vitamins and NHL. Nonusers of alcohol had an elevated NHL risk compared with users, and alcohol did not modify other nutrient-NHL associations. Findings suggest that one-carbon nutrients, particularly vitamin B6 and methionine, may be protective against NHL.

Validation of an HPLC method for the determination of ciprofloxacin in human plasma.

A simple HPLC method with fluorescence detection of ciprofloxacin in human plasma was developed and validated. After protein precipitation, chromatographic separation of ciprofloxacin in plasma was achieved at 35 degrees C with a C18 column and acetonitrile-phosphate mixture, pH 3, as mobile phase. Quantitative determination was performed by fluorimetry after excitation at 278 nm. The method was specific and validated with a limit of quantification of 41 ng/ml. The intra- and inter-day coefficients of variation were between 0.5 and 6.6% and accuracy between -2.02 and 7.04%. Ciprofloxacin was stable in plasma for 40 days at -20 degrees C and after three freezing-thawing cycles. The method has been applied in a bioequivalence study of two formulation of 500 mg ciprofloxacin.

Photocycle of dried acid purple form of bacteriorhodopsin.

The photocycle of dried bacteriorhodopsin, pretreated in a 0.3 M HCl solution, was studied. Some properties of this dried sample resemble that of the acid purple suspension: the retinal conformation is mostly all-trans, 15-anti form, the spectrum of the sample is blue-shifted by 5 nm to 560 nm, and it has a truncated photocycle. After photoexcitation, a K-like red-shifted intermediate appears, which decays to the ground state through several intermediates with spectra between the K and the ground state. There are no other bacteriorhodopsin-like intermediates (L, M, N, O) present in the photocycle. The K to K' transition proceeds with an enthalpy decrease, whereas during all the following steps, the entropic energy of the system decreases. The electric response signal of the oriented sample has only negative components, which relaxes to zero. These suggest that the steps after intermediate K represent a relaxation process, during which the absorbed energy is dissipated and the protein returns to its original ground state. The initial charge separation on the retinal is followed by limited charge rearrangements in the protein, and later, all these relax. The decay times of the intermediates are strongly influenced by the humidity of the sample. Double-flash experiments proved that all the intermediates are directly driven back to the ground state. The study of the dried acid purple samples could help in understanding the fast primary processes of the protein function. It may also have importance in technical applications.

Structural changes in bacteriorhodopsin during the photocycle measured by time-resolved polarized Fourier transform infrared spectroscopy.

The structural changes in bacteriorhodopsin during the photocycle are investigated. Time resolved polarized infrared spectroscopy in combination with photoselection is used to determine the orientation and motion of certain structural units of the molecule: Asp-85, Asp-96, Asp-115, the Schiff base, and several amide I vibrations. The results are compared with recently published x-ray diffraction data with atomic resolution about conformational motions during the photocycle. The orientation of the measured vibrations are also calculated from the structure data, and based on the comparison of the values from the two techniques new information is obtained: several amide I bands in the infrared spectrum are assigned, and we can also identify the position of the proton in the protonated Asp residues.

Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis.

BACKGROUND AND AIMS: There is a significant relationship between inheritance of high transforming growth factor (TGF)-beta1 and angiotensinogen-producing genotypes and the development of progressive hepatic fibrosis in patients with chronic hepatitis C. In cardiac and renal fibrosis, TGF-beta1 production may be enhanced by angiotensin II, the principal effector molecule of the renin-angiotensin system. The aim of the present study was to determine the effects of the angiotensin-converting enzyme inhibitor, captopril, on the progression of hepatic fibrosis in the rat bile duct ligation model. METHODS: Rats were treated with captopril (100 mg. kg(-1). day(-1)) commencing 1 or 2 weeks after bile duct ligation. Animals with bile duct ligation only and sham-operated animals served as controls. Four weeks after bile duct ligation, indices of fibrosis were assessed. RESULTS: Captopril treatment significantly reduced hepatic hydroxyproline levels, mean fibrosis score, steady state messenger RNA levels of TGF-beta1 and procollagen alpha1(I), and matrix metalloproteinase 2 and 9 activity. CONCLUSIONS: Captopril significantly attenuates the progression of hepatic fibrosis in the rat bile duct ligation model, and its effectiveness should be studied in human chronic liver diseases associated with progressive fibrosis.

Formation of a new buried charge drives a large-amplitude protein quake in photoreceptor activation.

Photoactive yellow protein (PYP) is a eubacterial photoreceptor and a structural prototype of the PAS domain superfamily of receptor and regulatory proteins. We investigate the activation mechanism of PYP using time-resolved Fourier transform infrared (FTIR) spectroscopy. Our data provide structural, kinetic, and energetic evidence that the putative signaling state of PYP is formed during a large-amplitude protein quake that is driven by the formation of a new buried charge, COO(-) of the conserved Glu46, in a highly hydrophobic pocket at the active site. A protein quake is a process consisting of global conformational changes that are triggered and driven by a local structural "fault". We show that large, global structural changes take place after Glu46 ionization via intramolecular proton transfer to the anionic p-coumarate chromophore, and are suppressed by the absence of COO(-) formation in the E46Q mutant. Our results demonstrate the significance of buried charge formation in photoreceptor activation. This mechanism may serve as one of the general themes in activation of a range of receptor proteins. In addition, we report the results of time-resolved FTIR spectroscopy of PYP crystals. The direct comparison of time-resolved FTIR spectroscopic data of PYP in aqueous solution and in crystals reveals that the structure of the putative signaling state is not developed in P6(3) crystals. Therefore, when the structural developments during the functional process of a protein are experimentally determined to be very different in crystals and solutions, one must be cautious in drawing conclusions regarding the functional mechanism of proteins based on time-resolved X-ray crystallography.

Differences in risk factors, atherosclerosis, and cardiovascular disease between ethnic groups in Canada: the Study of Health Assessment and Risk in Ethnic groups (SHARE)

BACKGROUND: Cardiovascular disease rates vary greatly between ethnic groups in Canada. To establish whether this variation can be explained by differences in disease risk factors and subclinical atherosclerosis, we undertook a population-based study of three ethnic groups in Canada: South Asians, Chinese, and Europeans. METHODS: 985 participants were recruited from three cities (Hamilton, Toronto, and Edmonton) by stratified random sampling. Clinical cardiovascular disease was defined by history or electrocardiographic findings. Carotid atherosclerosis was measured with B-mode ultrasonography. Conventional (smoking, hypertension, diabetes, raised cholesterol) and novel risk factors (markers of a prothrombotic state) were measured. FINDINGS: Within each ethnic group and overall, the degree of carotid atherosclerosis was associated with a higher prevalence of cardiovascular disease. South Asians had the highest prevalence of this condition compared with Europeans and Chinese (11%, 5%, and 2%, respectively, p=0.0004). Despite this finding, Europeans had more atherosclerosis (mean of the maximum intimal medial thickness 0.75 [0.16] mm) than South Asians (0.72 [0.15] mm), and Chinese (0.69 [0.16] mm). South Asians had an increased prevalence of glucose intolerance, higher total and LDL cholesterol, higher triglycerides, and lower HDL cholesterol, and much greater abnormalities in novel risk factors including higher concentrations of fibrinogen, homocysteine, lipoprotein (a), and plasminogen activator inhibitor-1. INTERPRETATION: Although there are differences in conventional and novel risk factors between ethnic groups, this variation and the degree of atherosclerosis only partly explains the higher rates of cardiovascular disease among South Asians compared with Europeans and Chinese. The increased risk of cardiovascular events could be due to factors affecting plaque rupture, the interaction between prothrombotic factors and atherosclerosis, or as yet undiscovered risk factors.

Differences in risk factors, atherosclerosis and cardiovascular disease between ethnic groups in Canada: the study of health assessment and risk in ethnic groups (SHARE).

Cardiovascular disease rates vary greatly between ethnic groups in Canada. To establish whether this variation can be explained by differences in disease risk factors and subclinical atherosclerosis, we undertook a population-based study of three ethnic groups in Canada: South Asians, Chinese and Europeans. A total of 985 participants were recruited from three cities (Hamilton, Toronto and Edmonton) by stratified random sampling. Clinical cardiovascular disease was defined by history or electrocardiographic findings. Carotid atherosclerosis was measured with B-mode ultrasonography. Conventional (smoking, hypertension, diabetes, raised cholesterol) and novel risk factors (markers of a prothrombotic state) were measured. Within each ethnic group and overall, the degree of carotid atherosclerosis was associated with a higher prevalence of cardiovascular disease. South Asians had the highest prevalence of this condition compared with Europeans and Chinese (11%, 5% and 2%, respectively; p=0.0004). Despite this finding, Europeans had more atherosclerosis (mean of the maximum intimal medial thickness 0.75 [0.16] mm) than South Asians (0.72 [0.15] mm) and Chinese (0.69 [0.16] mm). South Asians had an increased prevalence of glucose intolerance, higher total and low-density lipoprotein cholesterol, higher triglycerides and lower high-density lipoprotein cholesterol, and much greater abnormalities in novel risk factors including higher concentrations of fibrinogen, homocysteine, lipoprotein(a), and plasminogen activator inhibitor-1. Although there are differences in conventional and novel risk factors between ethnic groups, this variation and the degree of atherosclerosis only partly explains the higher rates of cardiovascular disease among South Asians compared with Europeans and Chinese. The increased risk of cardiovascular events could be due to factors affecting plaque rupture, the interaction between prothrombotic factors and atherosclerosis, or as yet undiscovered risk factors.

Chloride ion binding to bacteriorhodopsin at low pH: an infrared spectroscopic study.

Bacteriorhodopsin (bR) and halorhodopsin (hR) are light-induced ion pumps in the cell membrane of Halobacterium salinarium. Under normal conditions bR is an outward proton transporter, whereas hR is an inward Cl- transporter. There is strong evidence that at very low pH and in the presence of Cl-, bR transports Cl- ions into the cell, similarly to hR. The chloride pumping activity of bR is connected to the so-called acid purple state. To account for the observed effects in bR a tentative complex counterion was suggested for the protonated Schiff base of the retinal chromophore. It would consist of three charged residues: Asp-85, Asp-212, and Arg-82. This quadruplet (including the Schiff base) would also serve as a Cl- binding site at low pH. We used Fourier transform infrared difference spectroscopy to study the structural changes during the transitions between the normal, acid blue, and acid purple states. Asp-85 and Asp-212 were shown to participate in the transitions. During the normal-to-acid blue transition, Asp-85 protonates. When the pH is further lowered in the presence of Cl-, Cl- binds and Asp-212 also protonates. The binding of Cl- and the protonation of Asp-212 occur simultaneously, but take place only when Asp-85 is already protonated. It is suggested that HCl is taken up in undissociated form in exchange for a neutral water molecule.

The Study of Health Assessment and Risk in Ethnic groups (SHARE): rationale and design. The SHARE Investigators.

The Study of Health Assessment and Risk in Ethnic groups (SHARE) is a study to determine the risk factors for atherosclerosis among three ethnic populations in Canada. Three hundred and thirty South Asian Canadian, 320 Chinese Canadian and 320 European Canadian men and women between 35 and 75 years of age are being randomly sampled from communities in Hamilton and Toronto, Ontario and Edmonton, Alberta for assessment of conventional (i.e., smoking, dyslipidemia, diabetes and hypertension) and emerging (i.e., candidate genes for atherosclerosis, homocysteine, fibrinolytic parameters, neurohormones, glucose intolerance, markers of infection, socioeconomic status, psychosocial status and diet) cardiovascular disease risk factors. Subclinical atherosclerosis is measured by quantitative B-mode ultrasonography of the carotid arteries, and other objective measures of vascular disease are a 12-lead electrocardiogram, a two-dimensional echocardiogram, ankle to arm blood pressure ratio and urine microalbumin concentration. The relationship between the conventional and emerging risk factors, and atherosclerosis, vascular disease and markers of end-organ damage will be evaluated between and within ethnic groups.