The quantum dot vs. organic dye conundrum for ratiometric FRET-based biosensors: which one would you chose?

Förster resonance energy transfer (FRET) is a widely used and ideal transduction modality for fluorescent based biosensors as it offers high signal to noise with a visibly detectable signal. While intense efforts are ongoing to improve the limit of detection and dynamic range of biosensors based on biomolecule optimization, the selection of and relative location of the dye remains understudied. Herein, we describe a combined experimental and computational study to systematically compare the nature of the dye, i.e., organic fluorophore (Cy5 or Texas Red) vs. inorganic nanoparticle (QD), and the position of the FRET donor or acceptor on the biomolecular components. Using a recently discovered transcription factor (TF)-deoxyribonucleic acid (DNA) biosensor for progesterone, we examine four different biosensor configurations and report the quantum yield, lifetime, FRET efficiency, IC50, and limit of detection. Fitting the computational models to the empirical data identifies key molecular parameters driving sensor performance in each biosensor configuration. Finally, we provide a set of design parameters to enable one to select the fluorophore system for future intermolecular biosensors using FRET-based conformational regulation in in vitro assays and new diagnostic devices.

An Allosteric Transcription Factor DNA-Binding Electrochemical Biosensor for Progesterone.

We describe an electrochemical strategy to transduce allosteric transcription factor (aTF) binding affinity to sense steroid hormones. Our approach utilizes square wave voltammetry to monitor changes in current output as a progesterone (PRG)-specific aTF (SRTF1) unbinds from the cognate DNA sequence in the presence of PRG. The sensor detects PRG in artificial urine samples with sufficient sensitivity suitable for clinical applications. Our results highlight the capability of using aTFs as the biorecognition elements to develop electrochemical point-of-care biosensors for the detection of small-molecule biomarkers and analytes.

Paper-Based Progesterone Sensor Using an Allosteric Transcription Factor.

Progesterone monitoring is an essential component of in vitro fertilization treatments and reproductive management of dairy cows. Gold-standard biosensors for progesterone monitoring rely on antibodies, which are expensive and difficult to procure. We have developed an alternative transcription factor-based sensor that is superior to conventional progesterone biosensors. Here, we incorporate this transcription factor-based progesterone sensor into an affordable, portable paperfluidic format to facilitate widespread implementation of progesterone monitoring at the point of care. Oligonucleotides labeled with a fluorescent dye are immobilized onto nitrocellulose via a biotin-streptavidin interaction. In the absence of progesterone, these oligonucleotides form a complex with a transcription factor that is fluorescently labeled with tdTomato. In the presence of progesterone, the fluorescent transcription factor unbinds from the immobilized DNA, resulting in a decrease in tdTomato fluorescence. The limit of detection of our system is 27 nm, which is a clinically relevant level of progesterone. We demonstrate that transcription factor-based sensors can be incorporated into paperfluidic devices, thereby making them accessible to a broader population due to the portability and affordability of paper-based devices.

Implementing the Mindfulness-Based Interventions; Teaching Assessment Criteria (MBI:TAC) in Mindfulness-Based Teacher Training.

The Mindfulness-Based Interventions: Teaching Assessment Criteria (MBI:TAC) was originally developed as a tool to assess the teaching competence of mindfulness-based program (MBP) teachers. The tool was made freely available and has since been used by mindfulness-based teacher training organisations internationally. During this time the MBI:TAC has evolved in its usage, from an assessment tool to one which informally supports how MBP teachers are trained. In this article, we first examine the rationale for implementing the MBI:TAC in MBP teacher training; second, we offer practical guidance on ways of integrating the tool into teacher training pathways with awareness of its potential and possible pitfalls; and third, we offer guidance on using the tool as a framework for giving effective feedback to trainees on their teaching practice. Implementing the MBI:TAC in teacher training may support the quality and integrity of MBP teacher training, and thus ensure high quality MBP teachers graduating. In turn this may help avoid the 'implementation cliff' - that is, the quality of an intervention delivery is delivered in optimal conditions when it is being researched, and drops in quality when delivered in sub-optimal, 'real world' conditions.

Transcription Factor Based Small-Molecule Sensing with a Rapid Cell Phone Enabled Fluorescent Bead Assay.

Recently, allosteric transcription factors (TFs) were identified as a novel class of biorecognition elements for in vitro sensing, whereby an indicator of the differential binding affinity between a TF and its cognate DNA exhibits dose-dependent responsivity to an analyte. Described is a modular bead-based biosensor design that can be applied to such TF-DNA-analyte systems. DNA-functionalized beads enable efficient mixing and spatial separation, while TF-labeled semiconductor quantum dots serve as bright fluorescent indicators of the TF-DNA bound (on bead) and unbound states. The prototype sensor for derivatives of the antibiotic tetracycline exhibits nanomolar sensitivity with visual detection of bead fluorescence. Facile changes to the sensor enable sensor response tuning without necessitating changes to the biomolecular affinities. Assay components self-assemble, and readout by eye or digital camera is possible within 5 minutes of analyte addition, making sensor use facile, rapid, and instrument-free.

Hydrogel-Embedded Quantum Dot-Transcription Factor Sensors for Quantitative Progesterone Detection.

Immobilization of biosensors in or on a functional material is critical for subsequent device development and translation to wearable technology. Here, we present the development and assessment of an immobilized quantum dot-transcription factor-nucleic acid complex for progesterone detection as a first step toward such device integration. The sensor, composed of a polyhistidine-tagged transcription factor linked to a quantum dot and a fluorophore-modified cognate DNA, is embedded within a hydrogel as an immobilization matrix. The hydrogel is optically transparent, soft, and flexible as well as traps the quantum dot-transcription factor DNA assembly but allows free passage of the analyte, progesterone. Upon progesterone exposure, DNA dissociates from the quantum dot-transcription factor DNA assembly resulting in an attenuated ratiometric fluorescence output via Förster resonance energy transfer. The sensor performs in a dose-dependent manner with a limit of detection of 55 nM. Repeated analyte measurements are similarly successful. Our approach combines a systematically characterized hydrogel as an immobilization matrix and a transcription factor-DNA assembly as a recognition/transduction element, offering a promising framework for future biosensor devices.

Surface Immobilized Nucleic Acid-Transcription Factor Quantum Dots for Biosensing.

Immobilization of biosensors on surfaces is a key step toward development of devices for real-world applications. Here the preparation, characterization, and evaluation of a surface-bound transcription factor-nucleic acid complex for analyte detection as an alternative to conventional systems employing aptamers or antibodies are described. The sensor consists of a gold surface modified with thiolated Cy5 fluorophore-labeled DNA and an allosteric transcription factor (TetR) linked to a quantum dot (QD). Upon addition of anhydrotetracycline (aTc)-the analyte-the TetR-QDs release from the surface-bound DNA, resulting in loss of the Förster resonance energy transfer signal. The sensor responds in a dose-dependent manner over the relevant range of 0-200 µm aTc with a limit of detection of 80 nm. The fabrication of the sensor and the subsequent real-time quantitative measurements establish a framework for the design of future surface-bound, affinity-based biosensors using allosteric transcription factors for molecular recognition.

A Förster Resonance Energy Transfer-Based Ratiometric Sensor with the Allosteric Transcription Factor TetR.

A recent description of an antibody-free assay is significantly extended for small molecule analytes using allosteric transcription factors (aTFs) and Förster resonance energy transfer (FRET). The FRET signal indicates the differential binding of an aTF-DNA pair with a dose-dependent response to its effector molecule, i.e., the analyte. The new sensors described here, based on the well-characterized aTF TetR, demonstrate several new features of the assay approach: 1) the generalizability of the sensors to additional aTF-DNA-analyte systems, 2) sensitivity modulation through the choice of donor fluorophore (quantum dots or fluorescent proteins, FPs), and 3) sensor tuning using aTF variants with differing aTF-DNA binding affinities. While all of these modular sensors self-assemble, the design reported here based on a recombinant aTF-FP chimera with commercially available dye-labeled DNA uses readily accessible sensor components to facilitate easy adoption of the sensing approach by the broader community.

A progesterone biosensor derived from microbial screening.

Bacteria are an enormous and largely untapped reservoir of biosensing proteins. We describe an approach to identify and isolate bacterial allosteric transcription factors (aTFs) that recognize a target analyte and to develop these TFs into biosensor devices. Our approach utilizes a combination of genomic screens and functional assays to identify and isolate biosensing TFs, and a quantum-dot Förster Resonance Energy Transfer (FRET) strategy for transducing analyte recognition into real-time quantitative measurements. We use this approach to identify a progesterone-sensing bacterial aTF and to develop this TF into an optical sensor for progesterone. The sensor detects progesterone in artificial urine with sufficient sensitivity and specificity for clinical use, while being compatible with an inexpensive and portable electronic reader for point-of-care applications. Our results provide proof-of-concept for a paradigm of microbially-derived biosensors adaptable to inexpensive, real-time sensor devices.

Thought-Action Fusion in Individuals with a History of Recurrent Depression and Suicidal Depression: Findings from a Community Sample.

Although suicidal ideation is one of the most consistent symptoms across recurrent episodes of depression, the mechanisms underpinning its maintenance are poorly understood. In order to develop effective treatments for suicidally depressed patients, understanding what maintains suicidal distress is critical. We hypothesised that Thought-Action Fusion (TAF), i.e., to assume that having a thought has real world consequences, originally described in Obsessive-Compulsive Disorder, might be a bias in recurrently suicidally depressed people. To assess this, we revised the original TAF scale, and assessed TAF in three samples: healthy controls, recurrently depressed individuals with no history of suicidality (D-NS) and individuals with a history of recurrent suicidal depression (D-S). Exploratory and confirmatory factor analyses indicated a three-factor solution of TAF: (1) TAF for uncontrollable events, (2) self-suicidal TAF for suicidal acts related to oneself, and (3) TAF for positive controllable events. Compared to healthy controls, the D-NS group reported significantly higher total TAF, TAF uncontrollable, and TAF self-suicidal subscales, whilst positive controllable TAF was lower compared to healthy controls. Both D-S and D-NS samples reported higher TAF for suicidal thought compared to healthy controls, i.e., believing that having suicidal thoughts means they will act on them, however in the context of low mood this became more pronounced for the D-S group. These findings suggest that targeting TAF both in suicidal and non-suicidal depression has merit.

Dynamic outcome prediction in a socio-demographically diverse population-based cohort of extremely preterm neonates.

OBJECTIVE: Accurate outcome prediction is crucial for counseling parents and providing individualized treatment to extremely premature infants. We sought to improve upon existing prediction model by using a diverse population-based cohort of extremely premature live births (⩽28 weeks' gestation) for survival and survival without severe neonatal morbidity at different times throughout the first week of life and to evaluate potential differences by race/ethnicity and maternal education. STUDY DESIGN: Retrospective cohort study of all California live births from 2007 through 2011 with linked birth, death and hospital discharge records. RESULTS: A total of 6009 infants were included. In the validation data set at time of delivery, the area under the receiver-operating characteristic curve for the model containing all predictors was 0.863 for survival and 0.789 for survival without severe morbidity. The marginal probability of survival without severe neonatal morbidity of an Asian infant born to a mother with <12 years of education compared with the reference (Caucasian infant, mother with ⩾12 years of education) was -0.23 (95% confidence interval (CI) -0.31 to -0.15) for all infants at time of birth and -0.28 (95% CI -0.39 to -0.18) for infants with attempted resuscitation. Notably, no other differences by racial/ethnic category and maternal education emerged. CONCLUSIONS: Probabilities of survival and survival without major morbidity change rapidly throughout the first week of life. Extremely premature infants born to Asian mothers with less than a high school education appear to have a lower probability to survive without significant morbidity compared with their Caucasian peers.

Risk of preterm birth among women using drugs during pregnancy with elevated α-fetoprotein.

OBJECTIVE: Examine the risk of preterm birth (PTB) among women who use drugs during pregnancy and have elevated α-fetoprotein (AFP). STUDY DESIGN: The sample included California singleton live births in 2005 to 2010 contained within a hospital discharge database linked to the Prenatal Screening Program. A selection of mothers who did not use drugs was selected at a ratio of 4:1. Risk of PTB was calculated using adjusted odds ratios and 95% confidence intervals (CIs) for women who did or did not use drugs by their AFP percentile. RESULTS: We identified 7190 women who used drugs and selected 28 760 women who did not. Of women using cocaine with AFP ⩾95th percentile, 43.8% delivered prematurely. Women using drugs with AFP ⩾95th percentile were 11 to 35 times as likely to deliver <32 weeks. CONCLUSION: The combination of drug use and elevated AFP results in high rates of PTB. This combination results in an additive risk.

Population-based risks of mortality and preterm morbidity by gestational age and birth weight.

OBJECTIVE: The objective of this study is to examine the effect of small or large for gestational age (SGA/LGA) status on mortality and morbidity by gestational age. STUDY DESIGN: Logistic binomial regression was used to calculate relative risks (RRs) and 95% confidence intervals for infant mortality and preterm morbidities for SGA or LGA compared with appropriately grown (AGA) deliveries stratified by gestational age group. RESULTS: Compared with AGA infants of similar gestational age, SGA infants were at increased risk for infant mortality. Mortality risk was decreased for LGA infants born between 25 and 27 weeks (RR: 0.6) but increased for LGA infants born between 28 and 31 weeks (RR: 1.9). Risk of preterm morbidity was increased for SGA infants born between 28 and 38 weeks, but decreased for LGA infants born before 37 weeks. CONCLUSION: This study demonstrates the importance of considering birth weight for gestational age when evaluating morbidity and mortality risks.

Interpregnancy interval after live birth or pregnancy termination and estimated risk of preterm birth: a retrospective cohort study.

OBJECTIVES: We assessed whether interpregnancy interval (IPI) length after live birth and after pregnancy termination was associated with preterm birth (PTB). DESIGN: Multiyear birth cohort. SETTINGS: Fetal death, birth and infant death certificates in California merged with Office of Statewide Health Planning and Development. POPULATION: One million California live births (2007-10) after live birth and after pregnancy termination. METHODS: Logistic regression was used to estimate odds ratios (ORs) of PTB of 20-36 weeks of gestation and its subcategories for IPIs after a live birth and after a pregnancy termination. We used conditional logistic regression (two IPIs/mother) to investigate associations within mothers. MAIN OUTCOME MEASURE: PTB relative to gestations of ≥ 37 weeks. RESULTS: Analyses included 971 211 women with IPI after live birth, and 138 405 women with IPI after pregnancy termination with 30.6% and 74.6% having intervals of <18 months, respectively. IPIs of <6 months or 6-11 months after live birth showed increased odds of PTB adjusted ORs for PTB of 1.71 (95% CI 1.65-1.78) and 1.20 (95% CI 1.16-1.24), respectively compared with intervals of 18-23 months. An IPI >36 months (versus 18-23 months) was associated with increased odds for PTB. Short IPI after pregnancy termination showed a decreased OR of 0.87 (95% CI 0.81-0.94). The within-mother analysis showed the association of increased odds of PTB for short IPI, but not for long IPI. CONCLUSIONS: Women with IPI <1 or >3 years after a live birth were at increased odds of PTB-an important group for intervention to reduce PTB. Short IPI after pregnancy termination was associated with reduced odds for PTB and needs to be further explored. TWEETABLE ABSTRACT: Short and long IPI after live birth, but not after pregnancy termination, showed increased odds for PTB.

Outcomes of pregnancies with more than one positive prenatal screening result in the first or second trimester.

OBJECTIVE: To describe adverse outcomes and fetal abnormalities in women with a positive prenatal screening result for more than one disorder. STUDY DESIGN: Study participants were drawn from a population of 452 901 women pregnant with singletons entering the California Prenatal Screening Program in their first-trimester. Risk assessment was provided for trisomy 21 and trisomy 18 in the first-trimester and trisomy 21, trisomy 18, neural tube defects, and Smith-Lemli-Opitz syndrome in the second-trimester. Inclusion in this study required positive screening for more than one of the screened conditions and a completed outcome of pregnancy survey. RESULTS: A total of 874 women met our study inclusion criteria. Over 25% of these pregnancies had a fetus with a chromosomal abnormality. Of the euploid pregnancies, 6.9% had a fetus with a major birth defect. Of the pregnancies with a fetus with neither a chromosomal abnormality nor a major birth defect, 9.3% ended in fetal demise. Overall, more than 50% of women with multiple positive screening results had either a fetus with a birth defect or a poor pregnancy outcome. CONCLUSION: Although it is rare to screen positive for more than one condition, such results indicate a very high risk for chromosomal abnormality, fetal demise, or structural abnormality.

Maternal characteristics and mid-pregnancy serum biomarkers as risk factors for subtypes of preterm birth.

OBJECTIVE: To examine the relationship between maternal characteristics, serum biomarkers and preterm birth (PTB) by spontaneous and medically indicated subtypes. DESIGN: Population-based cohort. SETTING: California, United States of America. POPULATION: From a total population of 1 004 039 live singleton births in 2009 and 2010, 841 665 pregnancies with linked birth certificate and hospital discharge records were included. METHODS: Characteristics were compared for term and preterm deliveries by PTB subtype using logistic regression and odds ratios adjusted for maternal characteristics and obstetric factors present in final stepwise models and 95% confidence intervals. First-trimester and second-trimester serum marker levels were analysed in a subset of 125 202 pregnancies with available first-trimester and second-trimester serum biomarker results. MAIN OUTCOME MEASURE: PTB by subtype. RESULTS: In fully adjusted models, ten characteristics and three serum biomarkers were associated with increased risk in each PTB subtype (Black race/ethnicity, pre-existing hypertension with and without pre-eclampsia, gestational hypertension with pre-eclampsia, pre-existing diabetes, anaemia, previous PTB, one or two or more previous caesarean section(s), interpregnancy interval ≥ 60 months, low first-trimester pregnancy-associated plasma protein A, high second-trimester α-fetoprotein, and high second-trimester dimeric inhibin A). These risks occurred in 51.6-86.2% of all pregnancies ending in PTB depending on subtype. The highest risk observed was for medically indicated PTB <32 weeks in women with pre-existing hypertension and pre-eclampsia (adjusted odds ratio 89.7, 95% CI 27.3-111.2). CONCLUSIONS: Our findings suggest a shared aetiology across PTB subtypes. These commonalities point to targets for further study and exploration of risk reduction strategies. TWEETABLE ABSTRACT: Findings suggest a shared aetiology across preterm birth subtypes. Patterns may inform risk reduction efforts.

Maternal serum markers, characteristics and morbidly adherent placenta in women with previa.

OBJECTIVE: To examine associations with morbidly adherent placenta (MAP) among women with placenta previa. STUDY DESIGN: Women with MAP (cases) and previa alone (controls) were identified from a cohort of 236,714 singleton pregnancies with both first and second trimester prenatal screening, and live birth and hospital discharge records; pregnancies with aneuploidies and neural tube or abdominal wall defects were excluded. Logistic binomial regression was used to compare cases with controls. RESULT: In all, 37 cases with MAP and 699 controls with previa alone were included. Risk for MAP was increased among multiparous women with pregnancy-associated plasma protein-A (PAPP-A) ⩾95th percentile (⩾2.63 multiple of the median (MoM); adjusted OR (aOR) 8.7, 95% confidence interval (CI) 2.8 to 27.4), maternal-serum alpha fetoprotein (MS-AFP) ⩾95th percentile (⩾1.79 MoM; aOR 2.8, 95% CI 1.0 to 8.0), and 1 and ⩾2 prior cesarean deliveries (CDs; aORs 4.4, 95% CI 1.5 to 13.6 and 18.4, 95% CI 5.9 to 57.5, respectively). CONCLUSION: Elevated PAPP-A, elevated MS-AFP and prior CDs are associated with MAP among women with previa.

Effect of storage temperature on quality of light and full-fat ice cream.

Ice cream quality is dependent on many factors including storage temperature. Currently, the industry standard for ice cream storage is -28.9 °C. Ice cream production costs may be decreased by increasing the temperature of the storage freezer, thus lowering energy costs. The first objective of this research was to evaluate the effect of 4 storage temperatures on the quality of commercial vanilla-flavored light and full-fat ice cream. Storage temperatures used were -45.6, -26.1, and -23.3 °C for the 3 treatments and -28.9 °C as the control or industry standard. Ice crystal sizes were analyzed by a cold-stage microscope and image analysis at 1, 19.5, and 39 wk of storage. Ice crystal size did not differ among the storage temperatures of light and full-fat ice creams at 19.5 or 39 wk. An increase in ice crystal size was observed between 19.5 and 39 wk for all storage temperatures except -45.6 °C. Coldness intensity, iciness, creaminess, and storage/stale off-flavor of the light and full-fat ice creams were evaluated at 39 wk of storage. Sensory evaluation indicated no difference among the different storage temperatures for light and full-fat ice creams. In a second study, light and full-fat ice creams were heat shocked by storing at -28.9 °C for 35 wk and then alternating between -23.3 and -12.2 °C every 24h for 4 wk. Heat-shocked ice creams were analyzed at 2 and 4 wk of storage for ice crystal size and were evaluated by the sensory panel. A difference in ice crystal size was observed for light and full-fat ice creams during heat-shock storage; however, sensory results indicated no differences. In summary, storage of light or full-fat vanilla-flavored ice creams at the temperatures used within this research did not affect quality of the ice creams. Therefore, ice cream manufacturers could conserve energy by increasing the temperature of freezers from -28.9 to -26.1 °C. Because freezers will typically fluctuate from the set temperature, usage of -26.1 °C allows for a safety factor, even though storage at -23.3 °C did not affect ice cream quality.