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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.

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.