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Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nanoparticle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration., Journal of Physics: Photonics, 5 (2), ISSN:2515-7647

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nanoparticle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration., Comment: 181 pages, 61 figures

OBJECTIVE The Environmental Determinants of Diabetes in the Young (TEDDY) study is uniquely capable of investigating age-specific differences associated with type 1 diabetes. Because age is a primary driver of heterogeneity in type 1 diabetes, we sought to characterize by age metabolic derangements prior to diagnosis and clinical features associated with diabetic ketoacidosis (DKA). RESEARCH DESIGN AND METHODS The 379 TEDDY children who developed type 1 diabetes were grouped by age at onset (0–4, 5–9, and 10–14 years; n = 142, 151, and 86, respectively) with comparisons of autoantibody profiles, HLAs, family history of diabetes, presence of DKA, symptomatology at onset, and adherence to TEDDY protocol. Time-varying analysis compared those with oral glucose tolerance test data with TEDDY children who did not progress to diabetes. RESULTS Increasing fasting glucose (hazard ratio [HR] 1.09 [95% CI 1.04–1.14]; P = 0.0003), stimulated glucose (HR 1.50 [1.42–1.59]; P < 0.0001), fasting insulin (HR 0.89 [0.83–0.95]; P = 0.0009), and glucose-to-insulin ratio (HR 1.29 [1.16–1.43]; P < 0.0001) were associated with risk of progression to type 1 diabetes. Younger children had fewer autoantibodies with more symptoms at diagnosis. Twenty-three children (6.1%) had DKA at onset, only 1 (0.97%) of 103 with and 22 (8.0%) of 276 children without a first-degree relative (FDR) with type 1 diabetes (P = 0.008). Children with DKA were more likely to be nonadherent to study protocol (P = 0.047), with longer duration between their last TEDDY evaluation and diagnosis (median 10.2 vs. 2.0 months without DKA; P < 0.001). CONCLUSIONS DKA at onset in TEDDY is uncommon, especially for FDRs. For those without familial risk, metabolic monitoring continues to provide a primary benefit of reduced DKA but requires regular follow-up. Clinical and laboratory features vary by age at onset, adding to the heterogeneity of type 1 diabetes.

We report analytical expressions for optical forces acting on particles inside waveguides. The analysis builds on our previously reported Fourier Transform method to obtain Beam Shape Coefficients for any beam. Here we develop analytical expressions for the Beam Shape Coefficients in cylindrical and rectangular metallic waveguides. The theory is valid for particle radius a ranging from the Rayleigh regime to large microparticles, such as aerosols like virus loaded droplets. The theory is used to investigate how optical forces within hollow waveguides can be used to sort particles in “optical chromatography” experiments in which particles are optically propelled along a hollow-core waveguide. For Rayleigh particles, the axial force is found to scale with a6, while the radial force, which prevents particles from crashing into the waveguide walls, scales with a3. For microparticles, narrow Mie resonances create a strong wavelength dependence of the optical force, enabling more selective sorting. Several beam parameters, such as power, wavelength, polarization state and waveguide modes can be tuned to optimize the sorting performance. The analysis focuses on cylindrical waveguides, where meter-long liquid waveguides in the form of hollow-core photonic crystal fibers are readily available. The modes of such fibers are well-approximated by the cylindrical waveguide modes considered in the theory.

Domoic acid (DA) is a marine neurotoxin produced by several species of Pseudo-nitzschia. DA causes severe neurological toxicity in humans and animals. To address the current analytical need to quantify low levels of DA in human and animal body fluids, a sensitive and selective high performance liquid chromatography-tandem mass spectrometry method was developed to measure DA in plasma and urine. This method was fully validated to accurately and precisely quantify DA between 0.31 and 16 ng/mL in plasma and between 7.8 and 1000 ng/mL in urine. Our group introduced the use of a novel internal standard, tetrahydrodomoic acid to control for matrix effects and other sources of variability. This validated method will be useful to assess DA concentrations in biological samples of human or animal origin after suspected DA exposure from contaminated food. It will also be applicable to sentinel programs and research studies to analyze body fluids with low levels of DA.

Probiotics are linked to positive regulatory effects on the immune system. The aim of the study was to examine the association between the exposure of probiotics via dietary supplements or via infant formula by the age of 1 year and the development of celiac disease autoimmunity (CDA) and celiac disease among a cohort of 6520 genetically susceptible children. Use of probiotics during the first year of life was reported by 1460 children. Time-to-event analysis was used to examine the associations. Overall exposure of probiotics during the first year of life was not associated with either CDA (n = 1212) (HR 1.15; 95%CI 0.99, 1.35; p = 0.07) or celiac disease (n = 455) (HR 1.11; 95%CI 0.86, 1.43; p = 0.43) when adjusting for known risk factors. Intake of probiotic dietary supplements, however, was associated with a slightly increased risk of CDA (HR 1.18; 95%CI 1.00, 1.40; p = 0.043) compared to children who did not get probiotics. It was concluded that the overall exposure of probiotics during the first year of life was not associated with CDA or celiac disease in children at genetic risk.

OBJECTIVE β-Cell autoantibodies are a feature of the preclinical phase of type 1 diabetes. Here, we asked how frequently they revert in a cohort of children at risk for type 1 diabetes and whether reversion has any effect on type 1 diabetes risk. RESEARCH DESIGN AND METHODS Children were up to 10 years of age and screened more than once for insulin autoantibody, GAD antibody, and insulinoma antigen-2 antibodies. Persistent autoantibody was defined as an autoantibody present on two or more consecutive visits and confirmed in two reference laboratories. Reversion was defined as two or more consecutive negative visits after persistence. Time-dependent Cox regression was used to examine how reversion modified the risk of development of multiple autoantibodies and type 1 diabetes. RESULTS Reversion was relatively frequent for autoantibodies to GAD65 (19%) and insulin (29%), but was largely restricted to children who had single autoantibodies (24%) and rare in children who had developed multiple autoantibodies ( CONCLUSIONS Type 1 diabetes risk remained high in children who had developed multiple β-cell autoantibodies even when individual autoantibodies reverted. We suggest that monitoring children with single autoantibodies for at least 1 year after seroconversion is beneficial for stratification of type 1 diabetes risk.

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics studies of large sample cohorts can easily require from months to years to complete. Acquiring consistent, high-quality data in such large-scale studies is challenging because of normal variations in instrumentation performance over time, as well as artifacts introduced by the samples themselves, such as those because of collection, storage and processing. Existing quality control methods for proteomics data primarily focus on post-hoc analysis to remove low-quality data that would degrade downstream statistics; they are not designed to evaluate the data in near real-time, which would allow for interventions as soon as deviations in data quality are detected. In addition to flagging analyses that demonstrate outlier behavior, evaluating how the data structure changes over time can aide in understanding typical instrument performance or identify issues such as a degradation in data quality because of the need for instrument cleaning and/or re-calibration. To address this gap for proteomics, we developed Quality Control Analysis in Real-Time (QC-ART), a tool for evaluating data as they are acquired to dynamically flag potential issues with instrument performance or sample quality. QC-ART has similar accuracy as standard post-hoc analysis methods with the additional benefit of real-time analysis. We demonstrate the utility and performance of QC-ART in identifying deviations in data quality because of both instrument and sample issues in near real-time for LC-MS-based plasma proteomics analyses of a sample subset of The Environmental Determinants of Diabetes in the Young cohort. We also present a case where QC-ART facilitated the identification of oxidative modifications, which are often underappreciated in proteomic experiments.

Vitamin K is an umbrella term describing a family of molecules containing the 2-methyl-1,4,-naphthoquinone core. The most widely studied are phylloquinone (Vitamin K1 and a form of Vitamin K2, menaquinone-4 (MK-4), that differ in the degree of unsaturation along the C20-phytyl chain (Scheme 1). Whereas Vitamin K1 primarily functions as a regulator of hemostasis, MK-4 appears important for bone health;1 it is also implicated in vascular calcification2 and it regulates ATP production in mitochondria.3 While the physiological roles of Vitamin K continue to be evaluated, little information regarding metabolism and excretion of Vitamin K metabolites is available. In humans, Vitamin K1 and MK-4 are metabolized to two glucuronide conjugates of chain-shortened carboxylic acid metabolites, referred to as vitamin K acid 1 (1) and vitamin K acid 2 (2) (Figure 1).4–6 In order to quantify Vitamin K acid metabolites in biological matrices to understand Vitamin K metabolism, authentic analytical standards are required. Scheme 1 Terminal oxidation products of vitamin K metabolism Reported syntheses of vitamin K acid 1 (1) are incompletely described and required several laborious steps, some of which result in the partial isomerization of the side chain 2’,3’-double bond.7–10 The menadione-cyclopentadiene adduct 3 previously alkylated with C20-phytyl allylic halides11 to obtain Vitamin K1 was used as starting material for 1 in our synthesis (Scheme 2). Alkylation with geranyl bromide gave trans-geranyl adduct 4 in 79% yield. The cyclopentadiene protecting group was removed by heating 4 in AcOH with catalytic dodecyl-trimethylammonium bromide to produce 5 in 97% yield. The previously reported regioselective epoxidation of geraniol derivatives, followed by oxidative cleavage with periodic acid to yield corresponding aldehydes12 was utilized to obtain aldehyde 7. Epoxidation (mCPBA) of alkene 5 afforded 6 (67% yield), and subsequent periodic acid oxidation gave aldehyde 7 (65% yield). Aldehyde 7 was oxidized with potassium peroxymonosulfate13 yielding trans-vitamin K acid 1 (1) (70% yield). The trans configuration of (1) was confirmed by a 2D-NOESY experiment (supplemental), which did not show a strong NOE cross-peak between the vinyl proton at C2’ and the vinyl methyl group at C3’. Scheme 2 Improved Synthesis of vitamin K acid 1 (1) Previously reported syntheses of vitamin K acid 2 (2) required several steps with poor yields of intermediates.7,10 New methodology was utilized to synthesize vitamin K acid 2 (2) more efficiently (Scheme 3). Menadione-cyclopentadiene adduct 3 was alkylated with dimethylallyl chloride to afford compound 8 in 82% yield. Following deprotection, 9 was subjected to allylic oxidation14 with SeO2 to give allylic alcohol 10 (57% yield). Subsequent reduction of the allylic alcohol by ruthenium-catalyzed transfer hydrogenation15 afforded saturated alcohol 11 in 17% yield. Multiple attempts at improving the percent conversion and yield of 11 were made by experimenting with several ruthenium catalysts. Initially, transfer hydrogenation was attempted with [{RuCl(µ-Cl)(η6-C6Me6)}2] and Cs2CO3, but the double bond remained intact as evidenced by the corresponding 1H NMR triplet signal at C3’. Subsequently, saturation of 10 with [Ru(cod)Cl2]n and potassium hydroxide16 resulted in a 1:1 mixture of 10 and 11 (34% yield). Lastly, we tried [{RuCl(µ-Cl)(η6-para-cymene)}2] with Cs2CO3 and the 1H NMR showed 70% conversion. After increasing the catalyst:base ratio to 25:50 mol%, respectively, pure 11 was isolated in 17% yield. Naphthoquinone containing molecules such as Vitamin K and menadione are notoriously unstable when exposed to heat, light, alkali, and reducing conditions. In each of the transfer hydrogenation reactions attempted, greater than 60% of the starting material was not recovered, which we attribute to probable degradation of the naphthoquinone. To complete the synthesis, 11 was oxidized to the carboxylic acid with periodic acid and pyridinium chlorochromate17 to afford vitamin K acid 2 (2) (84% yield). Scheme 3 Improved Synthesis of vitamin K acid 2 (2) The syntheses described herein provide facile routes to vitamin K acid 1 and 2 utilizing established reaction procedures and inexpensive starting materials. Overall yields for vitamin K acid 1 and 2 were 23% and 5%, respectively. The limiting step in the vitamin K acid 2 synthesis is the saturation of 11, which is mainly the result of naphthoquinone instability under the experimental conditions described. During the synthesis of vitamin K acid 1, the trans configuration of the 2’,3’-double bond was retained resulting in the production all trans-vitamin K acid 1 without the need for geometric isomer recrystallization as previously described7. Overall, the synthesized metabolites will serve as authentic standards for our future investigations of vitamin K metabolism.

BACKGROUND: Diagnosis of type 1 diabetes often causes a negative psychological impact on families. We examined whether parents and children enrolled in The Environmental Determinants of Diabetes in the Young (TEDDY) study differ in their psychological adjustment to diabetes diagnosis compared to children diagnosed with diabetes in the community. METHODS: TEDDY follows 8676 children at genetic risk for type 1 diabetes from birth. Fifty-four TEDDY children diagnosed with diabetes and 54 age-matched community control children diagnosed with diabetes were enrolled. Participants were aged 3 to 10 years and study visits occurred at 3, 6, and 12 months postdiagnosis. Psychological measures included an adapted diabetes-specific State Anxiety Inventory, the Pediatric Quality of Life Inventory-Diabetes Module, and the Pediatric Inventory for Parents, which measures frequency and difficulty of parenting stress. RESULTS: A generalized estimating equation analysis based on a difference score between TEDDY children and community controls found no significant differences between TEDDY parents and community controls on parent diabetes-specific anxiety (P = .30). However, TEDDY children exhibited better diabetes-specific quality of life (P = .03) and TEDDY parents reported lower frequency (P = .004) and difficulty (P = .008) of parenting stress compared to community controls. CONCLUSIONS: Children diagnosed with at-risk for type 1 diabetes who have previously enrolled in research monitoring have improved diabetes quality of life and lower parenting stress postdiagnosis compared to children diagnosed in the community. Families in follow-up studies may be more prepared if their child is diagnosed with diabetes.

This article seeks to determine whether factors related to autoimmunity risk remain significant after the initiation of two or more diabetes-related autoantibodies and continue to contribute to type 1 diabetes (T1D) risk among autoantibody-positive children in The Environmental Determinants of Diabetes in the Young (TEDDY) study. Characteristics included are age at multiple autoantibody positivity, sex, selected high-risk HLA-DR-DQ genotypes, relationship to a family member with T1D, autoantibody at seroconversion, INS gene (rs1004446_A), and non-HLA gene polymorphisms identified by the Type 1 Diabetes Genetics Consortium (T1DGC). The risk of progression to T1D was not different among those with or without a family history of T1D (P = 0.39) or HLA-DR-DQ genotypes (P = 0.74). Age at developing multiple autoantibodies (hazard ratio = 0.96 per 1-month increase in age; 95% CI 0.95, 0.97; P < 0.001) and the type of first autoantibody (when more than a single autoantibody was the first-appearing indication of seroconversion [P = 0.006]) were statistically significant. Female sex was also a significant risk factor (P = 0.03). Three single nucleotide polymorphisms were associated with increased diabetes risk (rs10517086_A [P = 0.03], rs1534422_G [P = 0.006], and rs2327832_G [P = 0.03] in TNFAIP3) and one with decreased risk (rs1004446_A in INS [P = 0.006]). The TEDDY data suggest that non-HLA gene polymorphisms may play a different role in the initiation of autoimmunity than they do in progression to T1D once autoimmunity has appeared. The strength of these associations may be related to the age of the population and the high-risk HLA-DR-DQ subtypes studied.

Recent guidance on drug-drug interaction (DDI) testing recommends evaluation of circulating metabolites. However, there is little consensus on how to quantitatively predict and/or assess the risk of in vivo DDIs by multiple time-dependent inhibitors (TDIs) including metabolites from in vitro data. Fluoxetine was chosen as the model drug to evaluate the role of TDI metabolites in DDI prediction because it is a TDI of both CYP3A4 and CYP2C19 with a circulating N-dealkylated inhibitory metabolite, norfluoxetine. In pooled human liver microsomes, both enantiomers of fluoxetine and norfluoxetine were TDIs of CYP2C19, (S)-norfluoxetine was the most potent inhibitor with time-dependent inhibition affinity constant (KI) of 7 μM, and apparent maximum time-dependent inhibition rate (k(inact,app)) of 0.059 min(-1). Only (S)-fluoxetine and (R)-norfluoxetine were TDIs of CYP3A4, with (R)-norfluoxetine being the most potent (K(I) = 8 μM, and k(inact,app) = 0.011 min(-1)). Based on in-vitro-to-in-vivo predictions, (S)-norfluoxetine plays the most important role in in vivo CYP2C19 DDIs, whereas (R)-norfluoxetine is most important in CYP3A4 DDIs. Comparison of two multiple TDI prediction models demonstrated significant differences between them in in-vitro-to-in-vitro predictions but not in in-vitro-to-in-vivo predictions. Inclusion of all four inhibitors predicted an in vivo decrease in CYP2C19 (95%) and CYP3A4 (60-62%) activity. The results of this study suggest that adequate worst-case risk assessment for in vivo DDIs by multiple TDI systems can be achieved by incorporating time-dependent inhibition by both parent and metabolite via simple addition of the in vivo time-dependent inhibition rate/cytochrome P450 degradation rate constant (λ/k(deg)) values, but quantitative DDI predictions will require a more thorough understanding of TDI mechanisms.

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the β-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.

Bupropion is a widely used antidepressant and the recommended CYP2B6 probe drug. However, current understanding of bupropion elimination pathways is limited. Bupropion has three active circulating metabolites, OH-bupropion, threohydrobupropion, and erythrohydrobupropion, but together with bupropion these metabolites and their conjugates in urine represent only 23% of the dose, and the majority of the elimination pathways of bupropion result in uncharacterized metabolites. The aim of this study was to determine the structures of the uncharacterized bupropion metabolites using human clinical samples and in vitro incubations. Three new metabolites, 4'-OH-bupropion, erythro-4'-OH-hydrobupropion, and threo-4'-OH-hydrobupropion, were detected in human liver microsome incubations and were isolated from human urine. The structures of the metabolites were confirmed via comparison of UV absorbance, NMR spectra, and mass spectral data to those of the synthesized standards. In total, these metabolites represented 24% of the drug related material excreted in urine.

Itraconazole (ITZ) is a mixture of four cis-stereoisomers that inhibit CYP3A4 potently and coordinate CYP3A4 heme via the triazole nitrogen. However, (2R,4S,2'R)-ITZ and (2R,4S,2'S)-ITZ also undergo stereoselective sequential metabolism by CYP3A4 at a site distant from the triazole ring to 3'-OH-ITZ, keto-ITZ, and N-desalkyl-ITZ. This stereoselective metabolism demonstrates specific interactions of ITZ within the CYP3A4 active site. To further investigate this process, the binding and metabolism of the four trans-ITZ stereoisomers by CYP3A4 were characterized. All four trans-ITZ stereoisomers were tight binding inhibitors of CYP3A4-mediated midazolam hydroxylation (IC(50) 16-26 nM), and each gave a type II spectrum upon binding to CYP3A4. However, instead of formation of 3'-OH-ITZ, they were oxidized at the dioxolane ring, leading to ring scission and formation of two new metabolites of ITZ. These two metabolites were also formed from the four cis-ITZ stereoisomers, although not as efficiently. The catalytic rates of dioxolane ring scission were similar to the dissociation rates of ITZ stereoisomers from CYP3A4, suggesting that the heme iron is reduced while the triazole moiety coordinates to it and no dissociation of ITZ is necessary before catalysis. The triazole containing metabolite [1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone] also inhibited CYP3A4 (IC(50)15 μM) and showed type II binding with CYP3A4. The dioxolane ring scission appears to be clinically relevant because this metabolite was detected in urine samples from subjects that had been administered the mixture of cis-ITZ isomers. These data suggest that the dioxolane ring scission is a metabolic pathway for drugs that contain this moiety.

Three secondary amines desipramine (DES), (S)-fluoxetine [(S)-FLX], and N-desmethyldiltiazem (MA) undergo N-hydroxylation to the corresponding secondary hydroxylamines [N-hydroxydesipramine, (S)-N-hydroxyfluoxetine, and N-hydroxy-N-desmethyldiltiazem] by cytochromes P450 2C11, 2C19, and 3A4, respectively. The expected primary amine products, N-desmethyldesipramine, (S)-norfluoxetine, and N,N-didesmethyldiltiazem, are also observed. The formation of metabolic-intermediate (MI) complexes from these substrates and metabolites was examined. In each example, the initial rates of MI complex accumulation followed the order secondary hydroxylamine > secondary amine ≫ primary amine, suggesting that the primary amine metabolites do not contribute to formation of MI complexes from these secondary amines. Furthermore, the primary amine metabolites, which accumulate in incubations of the secondary amines, inhibit MI complex formation. Mass balance studies provided estimates of the product ratios of N-dealkylation to N-hydroxylation. The ratios were 2.9 (DES-CYP2C11), 3.6 [(S)-FLX-CYP2C19], and 0.8 (MA-CYP3A4), indicating that secondary hydroxylamines are significant metabolites of the P450-mediated metabolism of secondary alkyl amines. Parallel studies with N-methyl-d3-desipramine and CYP2C11 demonstrated significant isotopically sensitive switching from N-demethylation to N-hydroxylation. These findings demonstrate that the major pathway to MI complex formation from these secondary amines arises from N-hydroxylation rather than N-dealkylation and that the primary amines are significant competitive inhibitors of MI complex formation.

Retinoic acid (RA) is a critical signaling molecule that performs multiple functions required to maintain cellular viability. It is also used in the treatment of some cancers. Enzymes in the CYP26 family are thought to be responsible for the elimination of RA, and CYP26A1 appears to serve the most critical functions in this family. In spite of its importance, CYP26A1 has neither been heterologously expressed nor characterized kinetically. We expressed the rCYP26A1 in baculovirus-infected insect cells and purified the hexahistidine tagged protein to homogeneity. Heme incorporation was determined by carbon monoxide difference spectrum and a type 1 spectrum was observed with RA binding to CYP26A1. We found that RA is a tight binding ligand of CYP26A1 with low nM binding affinity. CYP26A1 oxidized RA efficiently (depletion K(m) 9.4+/-3.3nM and V(max) 11.3+/-4.3pmolesmin(-1)pmoleP450(-1)) when supplemented with P450 oxidoreductase and NADPH but was independent of cytochrome b5. 4-Hydroxy-RA (4-OH-RA) was the major metabolite produced by rCYP26A1 but two other primary products were also formed. 4-OH-RA was further metabolized by CYP26A1 to more polar metabolites and this sequential metabolism of RA occurred in part without 4-OH-RA leaving the active site of CYP26A1. The high efficiency of CYP26A1 in eliminating both RA and its potentially active metabolites supports the major role of this enzyme in regulating RA clearance in vivo. These results provide a biochemical framework for CYP26A1 function and offer insight into the role of CYP26A1 as a drug target as well as in fetal development and cell cycle regulation.

Our purpose is to bring attention to the antiepileptic properties of the Chinese herb tian ma and its constituents, as well as to suggest the potential for the development of new antiepileptic drugs (AEDs) related to this herb. All available literature regarding the chemistry, pharmacology, animal data, and clinical use of tian ma and its constituents are reviewed, showing that tian ma, its constituents, and its symbiotic fungus Armillaria mellea have antiepileptic properties in in vitro and in vivo models. One clinical study reportedly demonstrated the AED effects of a component of tian ma, vanillin. Thus, tian ma, its constituent vanillin, and its symbiotic fungus armillaria hold promise as cost-effective and less toxic alternatives to standard AEDs. In addition, similar chemical compounds may be developed as AEDs.

All-trans-retinoic acid (atRA), the active metabolite of vitamin A, induces gene transcription via binding to nuclear retinoic acid receptors (RARs). The primary hydroxylated metabolites formed from atRA by CYP26A1, and the subsequent metabolite 4-oxo-atRA, bind to RARs and potentially have biologic activity. Hence, CYP26A1, the main atRA hydroxylase, may function either to deplete bioactive retinoids or to form active metabolites. This study aimed to determine the role of CYP26A1 in modulating RAR activation via formation and elimination of active retinoids. After treatment of HepG2 cells with atRA, (4S)-OH-atRA, (4R)-OH-atRA, 4-oxo-atRA, and 18-OH-atRA, mRNAs of CYP26A1 and RARβ were increased 300- to 3000-fold, with 4-oxo-atRA and atRA being the most potent inducers. However, >60% of the 4-OH-atRA enantiomers were converted to 4-oxo-atRA in the first 12 hours of treatment, suggesting that the activity of the 4-OH-atRA was due to 4-oxo-atRA. In human hepatocytes, atRA, 4-OH-atRA, and 4-oxo-atRA induced CYP26A1 and 4-oxo-atRA formation was observed from 4-OH-atRA. In HepG2 cells, 4-oxo-atRA formation was observed even in the absence of CYP26A1 activity and this formation was not inhibited by ketoconazole. In human liver microsomes, 4-oxo-atRA formation was supported by NAD+, suggesting that 4-oxo-atRA formation is mediated by a microsomal alcohol dehydrogenase. Although 4-oxo-atRA was not formed by CYP26A1, it was depleted by CYP26A1 (Km = 63 nM and intrinsic clearance = 90 μl/min per pmol). Similarly, CYP26A1 depleted 18-OH-atRA and the 4-OH-atRA enantiomers. These data support the role of CYP26A1 to clear bioactive retinoids, and suggest that the enzyme forming active 4-oxo-atRA may be important in modulating retinoid action.

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D(3) metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23- and 24-hydroxylation of 1,25(OH)(2)D(3). No human microsomal cytochrome P450 enzyme tested, other than CYP3A5, supported these reactions. CYP3A4 exhibited opposite product stereochemical preference compared with that of CYP24A1, a known 1,25(OH)(2)D(3) hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH)(3)D(3), 1,24S,25(OH)(3)D(3), and 1,23S,25(OH)(3)D(3). Although the metabolic clearance of CYP3A4 was less than that of CYP24A1, comparison of metabolite profiles and experiments using CYP3A-specific inhibitors indicated that CYP3A4 was the dominant source of 1,25(OH)(2)D(3) 23- and 24-hydroxylase activity in both human small intestine and liver. Consistent with this observation, analysis of mRNA isolated from human intestine and liver (including samples from donors treated with phenytoin) revealed a general absence of CYP24A1 mRNA. In addition, expression of CYP3A4 mRNA in a panel of duodenal samples was significantly correlated with the mRNA level of a known vitamin D receptor gene target, calbindin-D9K. These and other data suggest that induction of CYP3A4-dependent 1,25(OH)(2)D(3) metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.

Itraconazole (ITZ) is a potent inhibitor of CYP3A in vivo. However, unbound plasma concentrations of ITZ are much lower than its reported in vitro Ki, and no clinically significant interactions would be expected based on a reversible mechanism of inhibition. The purpose of this study was to evaluate the reasons for the in vitro-in vivo discrepancy. The metabolism of ITZ by CYP3A4 was studied. Three metabolites were detected: hydroxy-itraconazole (OH-ITZ), a known in vivo metabolite of ITZ, and two new metabolites: keto-itraconazole (keto-ITZ) and N-desalkyl-itraconazole (ND-ITZ). OHITZ and keto-ITZ were also substrates of CYP3A4. Using a substrate depletion kinetic approach for parameter determination, ITZ exhibited an unbound K(m) of 3.9 nM and an intrinsic clearance (CLint) of 69.3 ml.min(-1).nmol CYP3A4(-1). The respective unbound Km values for OH-ITZ and keto-ITZ were 27 nM and 1.4 nM and the CLint values were 19.8 and 62.5 ml.min(-1).nmol CYP3A4(-1). Inhibition of CYP3A4 by ITZ, OH-ITZ, keto-ITZ, and ND-ITZ was evaluated using hydroxylation of midazolam as a probe reaction. Both ITZ and OH-ITZ were competitive inhibitors of CYP3A4, with unbound Ki (1.3 nM for ITZ and 14.4 nM for OH-ITZ) close to their respective Km. ITZ, OH-ITZ, keto-ITZ and ND-ITZ exhibited unbound IC50 values of 6.1 nM, 4.6 nM, 7.0 nM, and 0.4 nM, respectively, when coincubated with human liver microsomes and midazolam (substrate concentration < Km). These findings demonstrate that ITZ metabolites are as potent as or more potent CYP3A4 inhibitors than ITZ itself, and thus may contribute to the inhibition of CYP3A4 observed in vivo after ITZ dosing.

Propranolol, its 1"-mono-, di-, and trifluorinated analogs, and other related compounds were analyzed under electrospray ionization ion trap collision-induced dissociation (ESI-CID) and electron impact (EI) conditions. Interesting trends were observed in the fragment ions formed in both cases. Under ESI conditions, the abundances of product ions easily explained by protonation on the amine nitrogen decreased relative to the abundances of those formed from the ether-protonated species as the number of fluorines increased from zero to three. Under EI conditions, the distribution of fragment ions was shifted away from those arising from a nitrogen-centered cation radical and toward those arising from an ether oxygen-centered cation radical. The changes observed in apparent molecular sites of protonation and of ion radical formation in the mass spectra are consistent with the electron-withdrawing effects of the sequentially added fluorines. These effects are correlated with changes in solution phase pK( a)'s of the fluorinated amines.

Propranolol, deuterium- and 18O-labeled propranolol and related compounds were analyzed using an ion trap mass spectrometer equipped with a modified Finnigan API electrospray interface. Sequential product ion (MSn) experiments were used to elucidate fragmentation pathways for these compounds. The observed ions were compared to those observed under electron impact (EI) conditions. The electrospray ionization (ESI) ion trap spectra, as well as the EI spectra, afford useful information to allow assignments of most product ions, many of which retain portions of the aliphatic three-carbon side chain.

The E-isomer of 7-benzylidenenaltrexone (BNTX, la) was reported by Portoghese as a highly selective delta-opioid antagonist. The corresponding Z-isomer 1b was not readily available through direct aldol condensation of naltrexone (6) with benzaldehyde. Using the photochemical methods employed by Lewis to isomerize cinnamamides, we have obtained Z-isomer 1b in good yield from E-isomer 1a. A series of (E)- and (Z)-7-arylidenenaltrexone derivatives was prepared to study the effect of larger arylidene groups on opioid receptor affinity in this series. By aldol condensation of naltrexone (6) with benzaldehyde, 1-naphthaldehyde, 2-naphthaldehyde, 4-phenylbenzaldehyde, and 9-anthracaldehyde, the (E)-arylidenes were readily obtained. Photochemical isomerization afforded the corresponding Z-isomers. These compounds were evaluated via opioid receptor radioligand displacement assays. In these assays, the Z-isomers generally had higher affinity and were more delta-selective than the corresponding E-isomers. The (Z)-7-(1-naphthylidene)naltrexone (3b) showed the greatest selectivity (delta:mu ratio of 15) and highest affinity delta-binding (Ki = 0.7 nM). PM3 semiempirical geometry optimizations suggest a significant role for the orientation of the arylidene substituent in the binding affinity and delta-receptor selectivity. This work demonstrates that larger groups may be incorporated into the arylidene portion of the molecule with opioid receptor affinity being retained.

In this work, we show a simple experimental procedure to obtain CdTe/CdS (diameter=3–7 nm) core shell nanocrystals in colloidal suspension. The nanocrystals were characterized by transmission electronic microscopy and powder X-ray diffraction analysis. The optical properties were determined by electronic absorption, excitation and emission spectroscopies and are discussed in terms of chemical changes that occur at the surface of the particles.

Purpose. The oxidative metabolism of metoprolol was investigated in two human lymphoblastoma cell-lines transfected with variants of cDNA for cytochrome P4502D6.

Since 1908, when Mie reported analytical expressions for the fields scattered by a spherical particle upon incidence of plane-waves, generalizing his analysis for the case of an arbitrary incident wave has been an open question because of the cancellation of the prefactor radial spherical Bessel function. This cancellation was obtained before by our own group for a highly focused beam centered in the objective. In this work, however, we show for the first time how these terms can be canceled out for any arbitrary incident field that satisfies Maxwells equations, and obtain analytical expressions for the beam shape coefficients. We show several examples on how to use our method to obtain analytical beam shape coefficients for: Bessel beams, general hollow waveguide modes and specific geometries such as cylindrical and rectangular. Our method uses the vector potential, which shows the interesting characteristic of being gauge invariant. These results are highly relevant for speeding up numerical calculation of light scattering applications such as the radiation forces acting on spherical particles placed in an arbitrary electromagnetic field, as in an optical tweezers system.

A series of 6-O-ethers of 6 beta- and 6 alpha-naltrexol (6 and 7) were prepared to examine the effect of large aralkyl groups on affinity of the ligands for opioid receptors. The affinities of the 6 beta- and 6 alpha-O-ether with benzyl, biphenylmethyl, 1- and 2-naphthylmethyl, and 9-anthracylmethyl groups were determined. Preparation of the ligands was accomplished from suitably 3-O-protected derivatives of 6 and 7 by phase transfer catalyzed alkylation using aralkyl halides, followed by deprotection. Both 3-O-trityl and -benzyl protecting groups were used. In radioligand displacement assays, compounds from the 6 alpha-O ether series had higher affinity than the analogous diastereomers in the 6 beta-O series, with few exceptions. In the 6 alpha-O series, the benzyl ether (29) and the biphenylmethyl ether (30) had the highest affinity, similar to naltrexone. In the 6 beta-O series, the benzyl ether had the highest affinity. The larger aralkyl ethers had slightly less affinity. Large lipophilic 6 beta-O- and 6 alpha- O-aralkyl groups are readily accommodated in the drug-receptor interaction.

All-trans -retinoic acid ( at RA) is an important signaling molecule in all chordates. The cytochrome P450 enzymes CYP26 are believed to partially regulate cellular concentrations of at RA via oxidative metabolism and hence affect retinoid homeostasis and signaling. CYP26A1 and CYP26B1 are at RA hydroxylases that catalyze formation of similar metabolites in cell systems. However, they have only 40% sequence similarity suggesting differences between the two enzymes. The aim of this study was to determine whether CYP26A1 and CYP26B1 have similar catalytic activity, form different metabolites from at RA and are expressed in different tissues in adults. The mRNA expression of CYP26A1 and CYP26B1 correlated between human tissues except for human cerebellum in which CYP26B1 was the predominant CYP26 and liver in which CYP26A1 dominated. Quantification of CYP26A1 and CYP26B1 protein in human tissues was in agreement with the mRNA expression and showed correlation between the two isoforms. Qualitatively, recombinant CYP26A1 and CYP26B1 formed the same primary and sequential metabolites from at RA. Quantitatively, CYP26B1 had a lower K m (19 nM) and V max (0.8 pmol/min/pmol) than CYP26A1 ( K m = 50 nM and V max = 10 pmol/min/pmol) for formation of 4-OH-RA. The major at RA metabolites 4-OH-RA, 18-OH-RA and 4-oxo-RA were all substrates of CYP26A1 and CYP26B1, and CYP26A1 had a 2–10-fold higher catalytic activity towards all substrates tested. This study shows that CYP26A1 and CYP26B1 are qualitatively similar RA hydroxylases with overlapping expression profiles. CYP26A1 has higher catalytic activity than CYP26B1 and seems to be responsible for metabolism of at RA in tissues that function as a barrier for at RA exposure.

Female brownbanded cockroaches, Supella longipalpa, emit a sex pheromone that attracts males from a distance. This pheromone was isolated and identified as 5-(2,4-dimethylheptanyl)-3-methyl-2H-pyran-2-one (which we refer to as supellapyrone), and its structure was confirmed by synthesis. A racemic blend of the synthetic compound elicited behavioral and electrophysiological responses comparable to the natural pheromone across a range of doses. This compound is not only a very different type of cockroach pheromone but also makes up an additional class of natural products--namely, 3,5-dialkyl-substituted alpha-pyrones.

The objective of this study was to determine the pharmacokinetic parameters of clonidine during pregnancy compared with previously published data in nonpregnant subjects. Serial blood and urine samples were collected in 17 women during mid to late pregnancy over one steady-state dosing interval to determine clonidine noncompartmental pharmacokinetic parameters (n = 17) and creatinine clearance. In six of these pregnant subjects, maternal and umbilical cord (venous and arterial) plasma samples were collected at the time of delivery for measurement of clonidine concentrations. Clonidine apparent oral clearance was found to be 440 ± 168 ml/min during pregnancy compared with 245 ± 72 ml/min as previously reported in nonpregnant subjects (p < 0.0001) (Cunningham et al., 1994). There was a strong correlation (r = 0.82, p < 0.001) between clonidine renal clearance, adjusted for variation in glomerular filtration rate, and urine pH. Umbilical cord to maternal plasma clonidine concentration ratios were 1.0 ± 0.1 (arterial) and 1.0 ± 0.1 (venous). In conclusion, clonidine is cleared more rapidly in pregnant women than in nonpregnant subjects. At the time of delivery, the fetus is exposed to similar plasma clonidine concentrations as the mother.

Asymmetric synthesis of the diastereomeric 7-(1-hydroxyethyl)-2-[1-hydroxy-2-(tert-butylamino)ethyl]benzofurans (2), the benzylic hydroxylation metabolites of bufuralol (1), is described, and the absolute configurations of these diastereomers are assigned. 1"-Oxobufuralol (3) was reduced with a complex of (2S)-(-)-2-amino-3-methyl-1,1-diphenylbutan-1-ol and borane, yielding 2, which had a 95:5 ratio of the possible 1"R and 1"S isomers as determined by HPLC. Separation of the resulting diastereomers was facilitated by derivatization with the enantiomers of 1-phenethyl isocyanate (PEIC). The absolute configurations 1'S,1"R and 1'R,1"R were assigned to the diastereomers formed in excess, 2c and 2b, on the basis of the known stereochemistry of reduction of closely related alkyl phenyl ketones to R alcohols by using this chiral borane reagent. The circular dichroism spectra of the four isomeric benzylic alcohols were in agreement with these assignments. In the presence of the rat liver microsomal fraction, benzylic hydroxylation of bufuralol was significantly product stereoselective favoring formation of diastereomers with the 1"R absolute stereochemistry at the new chiral center in products from (1'R)-1 by a ratio of 4.5:1 [(1'R,1"R)-2:(1'R,1"S)-2] and by nearly 8:1 [(1'S,1"R)-2:(1'S,1"S)-2] from (1'S)-1. (1'R)-Bufuralol was more rapidly hydroxylated than was (1'S)-1, by about 3-fold. In the presence of human liver microsomes, (1'R)-bufuralol was also more rapidly hydroxylated than was (1'S)-1, by ca. 2.5-fold. However, product stereoselectivity from the 1'R enantiomer was reversed from that observed in the rat liver microsomal oxidation, with more (1"S)-carbinol being formed than 1"R isomer by nearly 4-fold. From (1'S)-1, about equal amounts of the two possible hydroxybufuralol diastereomers were formed. The results from the human liver microsomal studies are consistent with observed enantioselectivity of hydroxylation of bufuralol in vivo in humans.

Benzomorphans with an electrophilic group in the nitrogen substituent were prepared as potentially irreversible ligands for the kappa-opioid receptor. These were synthesized from products of the reaction of normetazocine with the enantiomers of 5-(iodomethyl)-gamma-butyrolactone (11). alpha-Methylene gamma-lactones 5 and 7 and endocyclic alpha,beta-unsaturated gamma-lactones 8 and 9 were prepared from the corresponding saturated gamma-lactones 13 and 23 possessing the "active" (1R,5R,9R)-benzomorphan stereochemistry. Only gamma-lactones 8, 9, 13, and 23, lacking the exocyclic methylene group, retain significant affinities for opioid receptor binding sites when compared with the reference compounds (2"S)-3 and (2"R)-3. As observed with these references compounds, greater binding affinity is also seen with gamma-lactone diastereomers having the 2"S stereochemistry in the nitrogen substituent. Although the gamma-lactones do not bind irreversibly in opioid receptor preparations, they do show kappa-receptor selectivities comparable to those observed for the reference compounds.

An improved laboratory scale synthesis of bufuralol (1) and 1″-oxobufuralol (4) was accomplished. The intermediate benzofurans were prepared via aromatization of 2,3-dihydrobenzofurans or by a one-step acidcatalyzed cyclization from 2,2-diethoxyethyl 4-bromo-6-ethyl-2-formylphenyl ether (23). Base-catalyzed cyclization of 3-(5-bromo-3-ethyl-2-hydroxyphenyl)-1.2-epoxypropane (16) provided the key intermediate, 5-bromo-7-ethyl-2-hydroxymethyl-2,3-dihydrobenzofuran (17). Selective functionalization of the C-2 and C-7 positions of the benzofuran ring system was accomplished to afford both 1 and 4.

Enantiomers of metoprolol containing six deuterium atoms in the isopropyl methyl groups were prepared in two steps from the sodium salt of 4-(2-methoxyethyl)phenol (3) and the commercially available (2R)- and (2S)-glycidyl 3-nitrobenzenesulfonates [(2R)-2 and (2S)-2]. The resulting (2R)- and (2S)-epoxides were opened using [2H6]-isopropylamine. The enantiomeric excesses were 93 and 95% for the deuterated (2R)- and (2S)-enantiomers of metoprolol [(2R)-1 and (2S)-1], respectively, as determined by chiral column HPLC.

Enantio- and diastereoselective aspects of oxidative metabolism of metoprolol ( 1 ) were examined in the presence of rat liver and human liver microsomes using a pseudoracemate of 1 , made up of equal molar (2R)- 1 - d 0 and (2S)- 1 - d 2 , as substrate. Both O-demethylation and α-hydroxylation showed only slight enantioselectivity, 2R 2S ratios being 1.18 and 0.93 for these pathways in rat liver microsomes and 1.09 and 0.92 in human liver microsomes. In the presence of the rat liver microsomal fraction, α-hydroxylation yielded predominantly the 1′ R -hydroxy product, 1′ R 1′S ratio > 12 , regardless of the stereochemistry of the side chain. In humans (extensive metabolizers) administered a single 50 mg oral dose of pseudoracemic metoprolol tartrate, urinary α-hydroxymetoprolol ( 2 ) accounted for 9.3 ± 2.4% of the dose, 2R 2S ratio of 0.85 ± 0.14 , and the carboxylic acid metabolite 4 , accounted for 52.7 ± 6.8% of the dose, 2R 2S ratio 1.15 ± 0.09. The data suggested that preferential O-demethylation of the (2 R )-enantiomer of 1 could contribute to the 2 S > 2 R plasma ratio of metoprolol enantiomers observed in this population.

2-(3,4-Dimethoxyphenyl)-2-isopropyl-5-[N-[4-(N-methyl-2-diazo- 3,3,3-trifluoropropionamido)phenethyl]methyl-amino]valeronitril e (3), a potential photoaffinity probe for Ca2+ channels related to verapamil (1), was prepared from N-methyl-4-nitrophenethylamine (7) and 2-(3,4-dimethoxyphenyl)-2-isopropyl-5-(methanesulfonoxy)valeron itrile (12). Compound 3 showed concentration-dependent negative inotropic effects in rat right myocardial ventricular strips, EC50 = (1.05 +/- 0.33) X 10(-7) M (mean +/- SD), being slightly less potent than gallopamil (2), EC50 = (2.18 +/- 0.66) X 10(-8) M. It displaced [3H]gallopamil in myocardial membranes, Ki = (3.76 +/- 1.55) X 10(-8) M, compared to 2, Ki = (1.55 +/- 0.16) X 10(-8) M. Photoactivation at 265 nm reduced the recoverable binding of [3H]gallopamil to 26% compared to no effect on 2. This agent may be a useful photoaffinity probe to aid in further characterization of Ca2+ channels.

Alpha- and beta-naltrexol derived esters 9 and 10 and ethers 11 and 12, each containing the alpha, beta-unsaturated ester functionality, were prepared as conformationally more flexible analogues of spiro-alpha-methylene-gamma-lactones 5 and 6. All were active in the opioid radioreceptor binding assay against [3H]bremazocine and more active against [3H]DAGO, indicating mu-subtype selectivity, but only ether 12 showed significant irreversible activity. We conclude that small structural changes, made in very closely related electrophilic opioids, lead to changes in receptor binding. All four compounds were long-acting antagonists to morphine in mice, with ester 10 being approximately equipotent with naltrexone.

Itraconazole (ITZ) is metabolized in vitro to three inhibitory metabolites: hydroxy-itraconazole (OH-ITZ), keto-itraconazole (keto-ITZ), and N-desalkyl-itraconazole (ND-ITZ). The goal of this study was to determine the contribution of these metabolites to drug-drug interactions caused by ITZ. Six healthy volunteers received 100 mg ITZ orally for 7 days, and pharmacokinetic analysis was conducted at days 1 and 7 of the study. The extent of CYP3A4 inhibition by ITZ and its metabolites was predicted using this data. ITZ, OH-ITZ, keto-ITZ, and ND-ITZ were detected in plasma samples of all volunteers. A 3.9-fold decrease in the hepatic intrinsic clearance of a CYP3A4 substrate was predicted using the average unbound steady-state concentrations (C(ss,ave,u)) and liver microsomal inhibition constants for ITZ, OH-ITZ, keto-ITZ, and ND-ITZ. Accounting for circulating metabolites of ITZ significantly improved the in vitro to in vivo extrapolation of CYP3A4 inhibition compared to a consideration of ITZ exposure alone.

The ability to observe quantitatively mechanical events in real time of biological phenomena is an important contribution of the Optical Tweezers technique for life sciences. The measurements of any mechanical property involves force measurements, usually performed using a microsphere as the force transducer. This makes the understanding of the photonic force theory critical. Only very sensitive and precise experimental 3D photonic force measurements for any particle size will be able to discriminate between different theoretical models. In particular it is important to obtain the whole photonic force curve as a function of the beam position instead of isolate particular points. We used a dual trap in an upright standard optical microscope, one to keep the particle at the equilibrium position and the other to disturb it. With this system we have been able to obtain these force curves as a function of x, y and z position, incident beam polarization and wavelength. We investigated the optical forces for wavelengths in and out of Mie resonances of dielectric microspherical cavities for both TM and TE modes and compared the experimental results with the calculations performed with different models for the optical force.

Partial wave decomposition of incident beams is the first task to be performed to impose boundary conditions at the particle interface in the calculation of the scattering of spherical particles. The coordinate's origin must be in the center of the particle and not at high symmetry positions of the beam. This can be a quite complicated problem, especially when a full vectorial diffraction description of the electromagnetic fields and highly focused laser beams are required where the paraxial limit fails. Traditional approximation techniques have been used to proceed forward and to obtain numerical results. The main fault relies on a radial dependence of the beam shape coefficients, which limits the validity of such approximations. Here we prove that the radial dependence will emerge from the solid angle integration in this way obtaining an exact, closed expression, without any approximation, for the beam shape coefficients, for an arbitrary beam shape, origin and polarization, the special case of a Gaussian beam is presented.

Since optical tweezers trapped microspheres can be used as an ultrasensitive force measurements technique, the knowledge of its theoretical description is of utmost importance. However, even the description of the incident electromagnetic fields under very tight focusing, typical of the optical trap, is not yet a closed problem. Therefore it is important to experimentally obtain whole accurate curves of the force as a function of wavelength, polarization and incident beam 3D position with respect to the center of the microsphere. Theoretical models for optical forces such as the Generalized Lorenz-Mie theory, can then be applied to the precisely evaluated experimental results. Using a dual trap in an upright standard optical microscope, one to keep the particle at the equilibrium position and the other to disturb it we have been able to obtain these force curves as a function of x, y and z position, incident beam polarization and also wavelength. Further investigation of optical forces was conducted for wavelengths in and out Mie resonances of the dielectric microspherical cavities for both TM and TE modes.

In the present work, PbTe quantum dots embedded in a dielectric host (SiO/sub 2/) were fabricated. The structural and optical properties of the multilayers were studied.