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Assays utilizing fluorophores are common throughout life science research and diagnostics, although detection limits are generally limited by weak emission intensity, thus requiring many labeled target molecules to combine their output to achieve higher signal-to-noise. We describe how the synergistic coupling of plasmonic and photonic modes can significantly boost the emission from fluorophores. By optimally matching the resonant modes of a plasmonic fluor (PF) nanoparticle and a photonic crystal (PC) with the absorption and emission spectrum of the fluorescent dye, a 52-fold improvement in signal intensity is observed, enabling individual PFs to be observed and digitally counted, where one PF tag represents one detected target molecule. The amplification can be attributed to the strong near-field enhancement due to the cavity-induced activation of the PF, PC band structure-mediated improvement in collection efficiency, and increased rate of spontaneous emission. The applicability of the method by dose-response characterization of a sandwich immunoassay for human interleukin-6, a biomarker used to assist diagnosis of cancer, inflammation, sepsis, and autoimmune disease is demonstrated. A limit of detection of 10 fg mL -1 and 100 fg mL -1 in buffer and human plasma respectively, is achieved, representing a capability nearly three orders of magnitude lower than standard immunoassays., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

Label-free detection and digital counting of nanometer-scaled objects such as nanoparticles, viruses, extracellular vesicles, and protein molecules enable a wide range of applications in cancer diagnostics, pathogen detection, and life science research. Here, we report the design, implementation, and characterization of a compact Photonic Resonator Interferometric Scattering Microscope (PRISM) designed for point-of-use environments and applications. The contrast of interferometric scattering microscopy is amplified through a photonic crystal surface, upon which scattered light from an object combines with illumination from a monochromatic source. The use of a photonic crystal substrate for interferemetric scattering microscopy results in reduced requirements for high-intensity lasers or oil-immersion objectives, thus opening a pathway toward instruments that are more suitable for environments outside the optics laboratory. The instrument incorporates two innovative elements that facilitate operation on a desktop in ordinary laboratory environments by users that do not have optics expertise. First, because scattering microscopes are extremely sensitive to vibration, we incorporated an inexpensive but effective solution of suspending the instrument's main components from a rigid metal framework using elastic bands, resulting in an average of 28.7 dBV reduction in vibration amplitude compared to an office desk. Second, an automated focusing module based on the principle of total internal reflection maintains the stability of image contrast over time and spatial position. In this work, we characterize the system's performance by measuring the contrast from gold nanoparticles with diameters in the 10-40 nm range and by observing various biological analytes, including HIV virus, SARS-CoV-2 virus, exosome, and ferritin protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nanoscale fluorescence emitters are efficient for measuring biomolecular interactions, but their utility for applications requiring single-unit observations is constrained by the need for large numerical aperture objectives, fluorescence intermittency, and poor photon collection efficiency resulting from omnidirectional emission. Photonic crystal (PC) structures hold promise to address the aforementioned challenges in fluorescence enhancement. In this review, we provide a broad overview of PCs by explaining their structures, design strategies, fabrication techniques, and sensing principles. Furthermore, we discuss recent applications of PC-enhanced fluorescence-based biosensors incorporated with emerging technologies, including nucleic acids sensing, protein detection, and steroid monitoring. Finally, we discuss current challenges associated with PC-enhanced fluorescence and provide an outlook for fluorescence enhancement with photonic-plasmonics coupling and their promise for point-of-care biosensing as well monitoring analytes of biological and environmental relevance. The review presents the transdisciplinary applications of PCs in the broad arena of fluorescence spectroscopy with broad applications in photo-plasmonics, life science research, materials chemistry, cancer diagnostics, and internet of things.

Label-free detection and digital counting of nanometer-scaled objects such as nanoparticles, viruses, extracellular vesicles, and protein molecules enable a wide range of applications in cancer diagnostics, pathogen detection, and life science research. The contrast of interferometric scattering microscopy is amplified through a photonic crystal surface, upon which scattered light from an object combines with illumination from a monochromatic plane wave source. The use of a photonic crystal substrate for interference scattering microscopy results in reduced requirements for high-intensity lasers or oil-immersion objectives, thus opening a pathway toward instruments that are more suitable for environments outside the optics laboratory. Here, we report the design, implementation, and characterization of a compact Photonic Resonator Interferometric Scattering Microscope (PRISM) designed for point-of-use environments and applications. The instrument incorporates two innovative elements that facilitate operation on a desktop in ordinary laboratory environments by users that do not have optics expertise. First, because scattering microscopes are extremely sensitive to vibration, we incorporated an inexpensive but effective solution of suspending the instrument's main components from a rigid metal framework using elastic bands, resulting in an average of 28.7 dBV reduction in vibration amplitude compared to an office desk. Second, an automated focusing module based on the principle of total internal reflection maintains the stability of image contrast over time and spatial position, facilitating automated data collection. In this work, we characterize the system's performance by measuring the contrast from gold nanoparticles with diameters in the 10-40 nm range and by observing various biological analytes, including HIV virus, SARS-CoV-2 virus, exosomes, and ferritin protein.

Several applications in health diagnostics, food, safety, and environmental monitoring require rapid, simple, selective, and quantitatively accurate viral load monitoring. Here, we introduce the first label-free biosensing method that rapidly detects and quantifies intact virus in human saliva with single-virion resolution. Using pseudotype SARS-CoV-2 as a representative target, we immobilize aptamers with the ability to differentiate active from inactive virions on a photonic crystal, where the virions are captured through affinity with the spike protein displayed on the outer surface. Once captured, the intrinsic scattering of the virions is amplified and detected through interferometric imaging. Our approach analyzes the motion trajectory of each captured virion, enabling highly selective recognition against nontarget virions, while providing a limit of detection of 1 × 10 3 copies/mL at room temperature. The approach offers an alternative to enzymatic amplification assays for point-of-collection diagnostics.

Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA's utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Unlike nearly all engineered materials which contain bonds that weaken under load, biological materials contain "catch" bonds which are reinforced under load. Consequently, materials, such as the cell cytoskeleton, can adapt their mechanical properties in response to their state of internal, non-equilibrium (active) stress. However, how large-scale material properties vary with the distance from equilibrium is unknown, as are the relative roles of active stress and binding kinetics in establishing this distance. Through course-grained molecular dynamics simulations, the effect of breaking of detailed balance by catch bonds on the accumulation and dissipation of energy within a model of the actomyosin cytoskeleton is explored. It is found that the extent to which detailed balance is broken uniquely determines a large-scale fluid-solid transition with characteristic time-reversal symmetries. The transition depends critically on the strength of the catch bond, suggesting that active stress is necessary but insufficient to mount an adaptive mechanical response., Competing Interests: Conflict of Interest The authors declare no conflict of interest.

Atomic force microscopy (AFM) enables determination of physical properties from single DNA molecules. Insertion of aromatic molecules into the structure of DNA results in morphological changes. However, the accompanying changes to elastic properties due to this insertion are not fully understood. AFM was used to examine the morphological effects of intercalator binding and report changes in the elastic properties of intrinsically straight DNA molecules. The persistence length and polymer extension were characterized in the presence of three intercalating molecules: ethidium bromide and the less well studied chloroquine and acridine. It was found that all three intercalators significantly increased the bending persistence length. In addition, an analysis of the normal bending modes of the static molecules corroborated these results. This approach of measuring binding effects of intercalators on DNA physical properties using a model system of intrinsically straight DNA is applicable to other DNA binding ligands and other modes of DNA interaction.

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-stranded DNA (ssDNA) intermediates, activating the DNA-damage response and handing off ssDNA to the appropriate downstream players. Six DNA-binding domains (DBDs) in RPA promote high-affinity binding to ssDNA yet also allow RPA displacement by lower affinity proteins. We generated fluorescent versions of Saccharomyces cerevisiae RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA while RPA remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. These findings reveal how RPA-interacting proteins with lower ssDNA binding affinities can access the occluded ssDNA and remodel individual DBDs to replace RPA.

Objective: Babor's A/B typology characterizes alcohol-dependence subtypes, which differ across multiple defining variables; however, differences in cigarette smoking and cessation between these subtypes have not been previously investigated. Topiramate reduces heavy drinking and has separately been found to help non-alcohol-dependent individuals quit smoking. This study tested the hypothesis that topiramate's effects on smoking would be moderated by alcohol-dependence subtype, and explored craving as a mediator of this response., Method: One hundred twenty-nine abstinent alcohol-dependent outpatient male smokers participated in this 12-week, randomized controlled trial comparing topiramate (maximum dosage 200 mg/day) with placebo, both with brief counseling, for smoking cessation. Participants were followed for 24 weeks following end of treatment., Results: Of the 125 participants with sufficient subtyping data, k-means cluster analysis categorized 52 (42%) as Type A alcoholics and 73 (58%) as Type B. Types A and B did not differ on baseline smoking characteristics, urges to smoke, or smoking consequence scores. Longitudinal mixed-effects regression indicated that the effect of treatment on smoking was moderated by the Type × Time interaction. Specifically, during the nontreatment follow-up phase, Type B's treated with topiramate had relative suppressed levels of smoking compared with placebo-treated Type B's. This moderating effect of the Type × Time interaction was mediated by intention to smoke and craving related to relief of negative affect., Conclusions: Type B alcoholics demonstrated suppressed levels of smoking in response to topiramate treatment as compared with placebo, but only during the nontreatment follow-up phase. This effect was mediated, in part, through intention to smoke and craving to smoke to relieve negative affect. Our findings extend other studies demonstrating a differential medication response by alcoholism subtype.

Background: Alcohol and nicotine dependence frequently co-occur, and quitting smoking might enhance long-term alcohol abstinence. Topiramate appears to help non-alcohol-dependent individuals quit smoking, and our pilot work suggested efficacy only in men. It also prevents relapse to alcohol in recently detoxified alcoholics. We evaluated topiramate in abstinent alcohol-dependent men to assess whether this medication (i) promotes smoking cessation and (ii) prevents alcohol and other drug relapse in the context of smoking cessation treatment., Methods: One hundred and twenty-nine alcohol-abstinent (mean ~6 months) alcohol-dependent male smokers (80% with other substance use disorders) participated in this 12-week randomized, double blind, parallel group comparison of topiramate (up to 200 mg/d) and placebo with a 24-week nontreatment follow-up period. The study was carried out sequentially at 2 academic centers in the Midwest and Southern California between March 23, 2009 and November 20, 2014. All participants received manual-guided smoking cessation counseling combined with medication-focused compliance enhancement therapy. Randomization was block designed by the research pharmacist in a 1:1 ratio. Participants, investigators, and research personnel were masked to treatment assignment. The primary smoking end point was biochemically confirmed 4-week continuous abstinence from smoking during weeks 9 to 12, while the secondary end point was relapse to any drinking or drug use during the entire 36-week evaluation period. Logistic regression was used to determine the effects of topiramate on quitting smoking and alcohol relapse, controlling for relevant covariates. The trial is registered at ClinicalTrials.gov (number NCT00802412) and is now closed., Results: Only a small proportion (7.9%) of topiramate-treated participants were able to quit smoking, and this cessation rate was similar to placebo (10.6%; odds ratio = 1.60; 95% confidence interval 0.4, 6.5; p = 0.51). Roughly 30% of the sample had a documented relapse to drinking or drug use during the study, and these rates were similar in the topiramate (20/63; 31.8%) and placebo groups (18/66; 27.3%; p = 0.58). Results of a longitudinal logistic regression model examining time to any alcohol relapse revealed no medication effect., Conclusions: Topiramate at a daily dosage of up to 200 mg per day, combined with smoking cessation and medication adherence counseling, had no effects on smoking cessation or the prevention of alcohol or drug relapse in male smokers who were in early or sustained full remission from alcohol and motivated to make a quit attempt. Alternative approaches for treating this high-risk, dually dependent population are needed., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Objective: The purpose of this study was to examine the presence and severity of eating disorder pathology in students representing South Africa's ethnically and culturally diverse population., Method: A questionnaire survey, which involved the Eating Attitude Test and the Bulimic Investigatory Test, was administered to 1,435 South African college students (739 Caucasian and 696 non-Caucasian) from six universities in two urban centers., Results: Black students scored significantly higher than the other ethnic groups on these measures. In addition, a comparable percentage of black and Caucasian female students had scores within the clinical range on these scales. Male students scored consistently lower than female students., Conclusions: This study's findings challenge the notion that eating disorders are primarily a Western, Caucasian phenomenon and raise the possibility that the risk of eating disorders may increase in developing societies.

Objective: To describe and evaluate a lay worker project designed to enhance the effective control of tuberculosis in a rural/farm setting., Design: A descriptive cohort study using both qualitative and quantitative research methodology., Setting and Participants: Farming community in the Klein Drakenstein area of Paarl, Western Cape, with health services provided by the Cape Metropolitan Council. The adherence of patients receiving tuberculosis treatment from 1 January 1993 to 31 December 1995 was analysed. The acceptability of the project was assessed using indepth interviews with key stakeholders., Results: A cohort of 402 tuberculosis patients was included in the study. This cohort was divided into the patients from farms that had participated in the project (intervention group) and those that had not (non-intervention group). The adherence data for children and adults were analysed separately. The adherence rates for children in both groups were the same (RR = 1.00, 95% Cl 0.88-1.14), whereas the adherence rate for the adult intervention group was significantly better than that of the non-intervention group (RR = 1.19, 95% Cl 1.08-1.31). The qualitative component of the investigation indicated a high level of commitment to the intervention from the members of the implementation team, the farm health workers and their employers. The farm health workers described how their role had assisted their communities and had improved their own perceptions of self-efficacy. The close bond that they had developed with the formal health sector had improved the access of the labourers on the farm to primary health care. Although the project had commenced as a tuberculosis initiative, the farm health workers were dealing with a variety of health issues. Their ongoing training was designed to facilitate this development. The formal health sector found their activities facilitated by the easy access that they had with a representative from each farm. The employers were satisfied that the project had increased the cost-effectiveness of their economic endeavours., Conclusion: This farm worker project appears to be a model of a well-designed and expertly managed community-based project for tuberculosis control in rural/farm areas.

The misuse and abuse of alcohol is widespread in South African society and is likely to have a large impact on the economy. A major burden is borne by the hospital care system, in particular the cost of alcohol-related trauma. The purpose of this study was to estimate the cost of alcohol misuse in terms of fatal and non-fatal trauma. The design of the study was a cross-sectional survey of key informants. Each key informant was mailed a list of 11 trauma categories found to be linked to alcohol in both the local and international literature. They were asked to make an estimate of the alcohol attributable fraction (AAF) for each trauma category, based on their clinical experience and knowledge of empirical research. The median estimates for each trauma category showed that alcohol misuse could be linked to a substantial amount of mortality and morbidity, particularly with respect to motor vehicle trauma and interpersonal violence. These AAF estimates were supported by the findings of other empirical research conducted in South Africa, as well as by the literature on mortality data in the U.S.A. This suggests that there is potential for a vast amount of injuries and deaths to be eliminated if alcohol misuse could be controlled or eliminated.

The infusion of methylprednisolone (MP) within 8 hours of injury for spinal cord injuries (SCI) has been advocated to improve the motor function of patients after this catastrophic injury. However, clinical improvement in the outcome of SCI has not been consistently identified, despite the use of MP. We reviewed the outcome of SCI patients with MP to those without MP (No-MP) at two Level I Trauma Centers from 1989-1992. Acute SCI patients were identified from the trauma registries with trauma demographics and hospital data obtained from registry and medical records. Rehabilitation data for Functional Independence Measure (FIM) was obtained from the rehabilitation institute database. Primary outcome parameters were mortality, and for survivors, patient mobility (6 point scale) and FIM scores. There were 145 acute SCI patients: 80 treated with MP and 65 with No-MP. FIM data was available on 45 MP and 25 No-MP patients. There was no difference in the admission trauma score, ICU length of stay (LOS), or hospital LOS between the two groups. The MP patients were significantly younger (30 years vs 38 years, P = < 0.05) and had lower ISS scores (24 vs 31, P = < 0.05). There was no statistically significant difference in mortality (MP, 3.8% vs No-MP, 10.7%) between the two groups. Although admission mobility was not statistically different (MP, 5.99 vs No-MP, 5.90), there was a significantly poorer discharge mobility in the MP group when compared to the No-MP group (MP, 5.16 vs No-MP, 4.67, P = < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

1. Providing optimal trauma care for large populations requires the collaboration and cooperation of hospitals providing all levels of care. 2. Those who are seriously injured or who have complex injuries requiring special resources are to be referred in a timely fashion to a trauma center capable of providing optimal care. 3. In two regional trauma networks, 24% of injured patients were referred to a trauma center for definitive care. Overall survival was excellent.

Competitive pressures to achieve a slim body shape may be of importance in the etiology of eating disorders in ballet dancers. This study examines the presence of anorexia nervosa-like symptoms in a group of 49 female ballet students (mean age = 18.9 years, SD +/- 1.9). All students were assessed for certain physical (weight and height) and psychological (Eating Attitude Test [EAT]) indices at the start of their academic training year. Thereafter, all subjects who presented with anorexia nervosa-like symptoms (EAT > or = 30, and/or with current secondary amenorrhea or primary amenorrhea if aged 16 years or over) at the initial assessment, were invited for a semistructured interview (Morgan-Russel scales) to determine their diagnostic status. Another aim of the study was to assess the prognostic implications of a diagnosis of anorexia nervosa in this sample. All subjects previously interviewed were invited for a follow-up assessment at 10 months. Anorexia nervosa could be diagnosed in 2 students (4.1%), whilst another 4 students (8.2%) presented with "partial syndrome" anorexia nervosa. All diagnosed students managed to complete their academic training year. The development and implications of a diagnosis of anorexia nervosa in the ballet students are discussed.

1. High-voltage electrophoresis and chromatography before and after reaction with 5-dimethylaminonaphthalene-1-sulphonyl chloride have identified putrescine and spermidine in hydrolysates of cyst coat proteins from the protozoan Colpoda steinii. 2. Amounts present varied with putrescine up to 19.7 and spermidine up to 16.9 residues per 1000 amino acid residues. 3. The amines were not, in the main, removed by acid or alkaline extraction or by reprecipitation. They were present in hydrolysates of peptides isolated electrophoretically from acid-degraded coat protein. 4. Proteolysis of oxidised coat protein produced a soluble core polypeptide to which the major proportion of the amines were attached and which had a simple composition. It was composed almost entirely of glutamic acid or glutamine, glycine, serine and cysteic acid, these residues being present in the approximate ratio of 10:2:1:1. 5. When coat protein was treated with 5-dimethylaminonaphthalene-1-sulphonyl chloride and hydrolysed no fully substituted amines could be detected but putrescine with one group substituted and spermidine derivatives with one and two groups substituted were present.