On the Behavior of Nanoparticles beyond the Nanopore Interface.

Recent advances in solid-state and biological nanopore sensors have produced a deluge of analytical techniques for in situ characterization of bio-nano colloidal dispersions; however, the transport forces governing particle movement into and out of the nanopore are not yet fully understood. Herein, we study the motion of particles outside the smaller opening of an elastomeric size-tunable nanopore and relate this motion to existing transport forces known to act on particles within the pore. Subsequently, we develop a combined optoelectronic approach which allows the comparison of both resistive pulse sensing and single particle tracking-based techniques for particle size characterization and, intriguingly, measurements of the ensemble particle motion induced by a combination of particle electrophoresis as well as pressure-driven and electroosmotic flows through the sensor nanopore. We find evidence suggesting that although bulk fluid flow from the pore tends to drive particle motion, in certain circumstances, electrophoretically driven motion can dominate bulk fluid flow-driven motion even at large distances from the pore opening. By permitting direct observation of the behavior of fluids at the nanopore interface, this approach enables a greater understanding of the transport forces acting on particles as they migrate toward and move through nanopore sensors-with implications for future particle characterization systems and for nanopore methods in general.

Long-term evaluation of the rise in legal age-of-sale of cigarettes from 16 to 18 in England: a trend analysis.

OBJECTIVE: To assess the long-term impact of the increase in age-of-sale of cigarettes from 16 to 18 in England in October 2007. METHODS: Data were collected between November 2006 and September 2018 on 252,601 participants taking part in a nationally representative survey of adults aged 16+ in England, the Smoking Toolkit Study (STS). We assessed the impact of the introduction of the increase in age-of-sale on prevalence of ever smoking, current smoking, and quit attempts, among 16-17 year olds compared with 18-24 year olds. RESULTS: Following the increase in age-of-sale, there was a declining trend in ever smoking that was greater among 16-17 year olds than 18-24 year olds (OR 0.990 versus OR 0.993; p = 0.019). Data on current smoking were insensitive to detect a difference between the age groups in a step-level change or change in trend following the increase in age-of-sale (Bayes factors (BFs) 0.75 and 2.10). Data on quit attempts were also insensitive to detect a change in trend (BF 0.71), and despite a greater step-level decline among those aged 16-17 (OR 0.311 versus OR 0.547, p = 0.025), quit attempts remained higher among those aged 16-17. Secondary analysis indicated that post-policy change, trends in current and ever smoking were linear for 16-17 year olds but quadratic for 18-24 year olds (slowing decline). CONCLUSION: There is some evidence from an assessment of long-term trends in the Smoking Toolkit Study that the increase in legal age-of-sale of cigarettes in England was associated with a greater long-term decline in ever smoking among those aged 16-17 compared with those aged 18-24.

How Do Treatment Priorities Differ Between Patients in HIV Care and Their Providers? A Mixed-Methods Study.

Evidence suggests priorities differ between patients in HIV care and their providers regarding topics most important to address in care. At five U.S. sites, we asked patients and providers to prioritize 25 potential topic areas to address during routine visits, and invited patients to discuss selection rationale. Patients (n = 206) and providers (n = 17) showed high discordance in rank order priorities (X2 (24, 223) = 71.12; p < 0.0001). Patients ranked social domains such as HIV stigma highly; a higher proportion of providers prioritized substance use domains. HIV stigma was a higher priority for patients in care fewer than 6 years (Fisher's exact p = 0.0062), nonwhite patients (Fisher's exact p = 0.0114), and younger patients (Fisher's exact p = 0.0281). Patients' priorities differed between men and women (X2 (24, 188) = 52.89; p < 0.0001), white race vs. other races (X2 (24, 206) = 48.32; p = 0.0023), and Latinos vs. non-Latinos (X2 (24, 206) = 48.65; p = 0.0021). Interviews (n = 79) revealed perceived impact of social context on health and health behaviors.

Isothermal Point Mutation Detection: Toward a First-Pass Screening Strategy for Multidrug-Resistant Tuberculosis.

Point mutations in DNA are useful biomarkers that can provide critical classification of disease for accurate diagnosis and to inform clinical decisions. Conventional approaches to detect point mutations are usually based on technologies such as real-time polymerase chain reaction (PCR) or DNA sequencing, which are typically slow and require expensive lab-based equipment. While rapid isothermal strategies such as recombinase polymerase amplification (RPA) have been proposed, they tend to suffer from poor specificity in discriminating point mutations. Herein, we describe a novel strategy that enabled exquisite point mutation discrimination with isothermal DNA amplification, using mismatched primers in conjunction with a two-round enrichment process. As a proof of concept, the method was applied to the rapid and specific identification of drug-resistant Mycobacterium tuberculosis using RPA under specific conditions. The assay requires just picogram levels of genomic DNA input, is sensitive and specific enough to detect 10% point mutation loading, and can discriminate between closely related mutant variants within 30 min. The assay was subsequently adapted onto a low-cost 3D-printed isothermal device with real-time analysis capabilities to demonstrate a potential point-of-care application. Finally, the generic applicability of the strategy was shown by detecting three other clinically important cancer-associated point mutations. We believe that our assay shows potential in a broad range of healthcare screening processes for detecting and categorizing disease phenotypes at the point of care, thus reducing unnecessary therapy and cost in these contexts.

Observations of Tunable Resistive Pulse Sensing for Exosome Analysis: Improving System Sensitivity and Stability.

Size distribution and concentration measurements of exosomes are essential when investigating their cellular function and uptake. Recently, a particle size distribution and concentration measurement platform known as tunable resistive pulse sensing (TRPS) has seen increased use for the characterization of exosome samples. TRPS measures the brief increase in electrical resistance (a resistive pulse) produced by individual submicrometer/nanoscale particles as they translocate through a size-tunable submicrometer/micrometer-sized pore, embedded in an elastic membrane. Unfortunately, TRPS measurements are susceptible to issues surrounding system stability, where the pore can become blocked by particles, and sensitivity issues, where particles are too small to be detected against the background noise of the system. Herein, we provide a comprehensive analysis of the parameters involved in TRPS exosome measurements and demonstrate the ability to improve system sensitivity and stability by the optimization of system parameters. We also provide the first analysis of system noise, sensitivity cutoff limits, and accuracy with respect to exosome measurements and offer an explicit definition of system sensitivity that indicates the smallest particle diameter that can be detected within the noise of the trans-membrane current. A comparison of exosome size measurements from both TRPS and cryo-electron microscopy is also provided, finding that a significant number of smaller exosomes fell below the detection limit of the TRPS platform and offering one potential insight as to why there is such large variability in the exosome size distribution reported in the literature. We believe the observations reported here may assist others in improving TRPS measurements for exosome samples and other submicrometer biological and nonbiological particles.

The perceptions of UK youth of branded and standardized, 'plain' cigarette packaging.

BACKGROUND: Tobacco packaging is an important form of promotion. Standardizing cigarette packages ('plain' packaging) represents a novel tobacco control policy. This study examined perceptions of branded and standardized cigarette packages among British youth. METHODS: Seven hundred twelve youth aged 11-17 completed an online survey. Participants viewed pairs of packages altered using a 3 × 2 factorial design: health warning type (40% text, 40% pictorial or 80% pictorial) × standardized pack colour (white vs. brown). A discrete-choice task was used in which participants selected packs based on attractiveness, taste, tar, health risk, impact of health warning and enticement to start smoking. Participants also compared regular Silk Cut and 'Superslims' Silk Cut packs. Participants completed a final selection task from two standardized and two branded packs. RESULTS: Warning type was significantly associated with all six outcomes: packs with larger pictorial warnings were more likely to be perceived as less attractive, less smooth, greater health risk, higher tar delivery, more effective health warnings and less likely to encourage initiation. The same pattern was found for brown vs. white standardized packages, with the exception of attractiveness and initiation. Compared with the regular Silk Cut pack, the 'Superslims' Silk Cut pack was perceived as significantly more favourable on all six outcomes. Finally, among respondents who selected a pack in the pack selection task, 95.1% selected a branded pack vs. 4.9% who selected a standardized pack. CONCLUSIONS: Increasing the size of pictorial health warnings and standardizing the appearance and shape of packages may discourage smoking initiation among young people.

Point-of-need mastitis pathogen biosensing in bovine milk: From academic sample preparation novelty to industry prototype field testing.

Bovine mastitis is an inflammation of the mammary gland, and it is the most common infectious disease in dairy cattle. Mastitis reduces milk yield and quality, costing dairy farmers millions of dollars each year. The aim of this study was to develop a point-of-need test for identifying mastitis pathogens that is field portable, cost-effective and can be used with minimal training. Using a proprietary polymer-based milk sample preparation method to rapidly extract pathogen DNA in milk samples, we demonstrated quantitative Polymerase Chain Reaction (qPCR) assays for six common bovine bacterial mastitis pathogens: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Mycoplasma bovis and Escherichia coli. We also implemented this sample preparation method on a prototype point-of-need system in a proof-of-concept field trial to evaluate user experience. Importantly, the protype system enabled a sample-to-result turnaround time of within 70 min to quantitatively detect all six target pathogens. The key advantage of our point-of-need prototype system is being culture-independent yet providing automated milk sample preparation for molecular identification of key mastitis pathogens by non-expert users. Our point-of-need prototype system showed a good correlation to laboratory-based qPCR for target pathogen detection outcomes, thus potentially removing the need for milk samples to be transported off-site for laboratory testing. Above all, we successfully achieved our objective of developing a point-of-need biosensor technology for mastitis and increased its readiness level with industry partners towards technology commercialization.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2µ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2µ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

Quantitative method for evaluation of aesthetic results after laser treatment for birthmarks.

Subjective assessment of results after laser treatment for birthmarks does not provide a validated method for clinicians. Previous reports concerning objective evaluation using L*a*b color coordinates were only partially successful due to difficulties in standardizing and comparing colors from pre- and posttreatment photographs. The study aimed to present a reliable and clinically applicable method of aesthetic result assessment after laser treatment for birthmarks. All 48 patients, included in the study, were treated for birthmarks on the face or neck using laser. Each pre- and posttreatment photograph was subjected to triple objective comparative assessment of color fading with use of a computer program, as well as to subjective evaluation by three core physicians and three laypeople. Objective analysis was based on an innovative method using combined L*a*b and hue saturation value color coordinates. Accuracy was higher between objective assessment with the computer program and subjective evaluation by core physicians than by laypeople. Repeatability of results was higher with the use of the computer program than among core physicians or among laypeople. In conclusion, our method may be considered for objective assessment of the results after laser treatment of vascular and pigmented birthmarks.

A variable pressure method for characterizing nanoparticle surface charge using pore sensors.

A novel method using resistive pulse sensors for electrokinetic surface charge measurements of nanoparticles is presented. This method involves recording the particle blockade rate while the pressure applied across a pore sensor is varied. This applied pressure acts in a direction which opposes transport due to the combination of electro-osmosis, electrophoresis, and inherent pressure. The blockade rate reaches a minimum when the velocity of nanoparticles in the vicinity of the pore approaches zero, and the forces on typical nanoparticles are in equilibrium. The pressure applied at this minimum rate can be used to calculate the zeta potential of the nanoparticles. The efficacy of this variable pressure method was demonstrated for a range of carboxylated 200 nm polystyrene nanoparticles with different surface charge densities. Results were of the same order as phase analysis light scattering (PALS) measurements. Unlike PALS results, the sequence of increasing zeta potential for different particle types agreed with conductometric titration.

Quantitative sizing of nano/microparticles with a tunable elastomeric pore sensor.

The use of a "size-tunable" polyurethane resistive pulse sensor for quantitative sizing of nano- and microparticles is presented. A linear relationship, as first suggested by Maxwell, between particle volume and change in electric resistance across the pore was observed. Particle sizes were quantified for a given size-tunable membrane, by first creating a linear calibration curve to a series of monodisperse carboxylated polystyrene particles of various diameters and then applying this curve to calculate the size of "unknown" nanoparticles. The diameters of a selection of synthetic and biological particles, being PMMA and nonfunctionalized polystyrene particles, along with biological nanoparticles (adenovirus) were calculated using this methodology. Calculated particle diameters and coefficients of variation were shown to be in good agreement with both transmission electron microscopy and dynamic light scattering results.

Who would be targeted by increasing the legal age of sale of cigarettes from 18 to 21? A cross-sectional study exploring the number and characteristics of smokers in England.

AIMS: To establish the number of smokers in England who would be targeted by increasing the age of sale of cigarettes from 18 to 21 years and to assess the smoking and socio-demographic profile of those smokers. DESIGN AND SETTING: Nationally representative cross-sectional survey of adults in England conducted between January 2009 and July 2019. PARTICIPANTS: A total of 219 720 adults. MEASUREMENTS: All participants reported their current smoking status and socio-demographic characteristics (i.e. age, gender, home ownership, social grade and ethnicity). Smokers reported motivation to quit, urges to smoke and the Heaviness of Smoking Index (HIS). Weighted prevalence statistics were calculated. Multinomial regression and logistic regression were used to assess differences in smoking characteristics among smokers and socio-demographic characteristics relative to non-smokers. FINDINGS: The prevalence of smoking between January 2009 and July 2019 was highest among those aged 21-30. In 2019, 15.6% [95% confidence interval (CI) = 12.8-18.8%] of 18-20-year-olds reported smoking, which is estimated to represent 364 000 individuals in England. Relative to smokers aged 18-20, older smokers (aged 21+) had a higher motivation to quit smoking [odds ratios (ORs) = 1.40-1.45 range] and higher nicotine dependency as measured by urges to smoke (ORs = 1.06-1.24 range) and HSI (ORs = 1.05-2.85 range). Compared with non-smokers aged 18-20, smokers in this age group had lower odds of being female (OR = 0.89) and higher odds of being of white ethnicity (OR = 2.78) and from social grades C1-E (lower social grades) compared with AB (higher social grades) (OR = 1.19-1.83 range). CONCLUSION: Increasing the age of sale of cigarettes to 21 years in England would currently target approximately 364 000 lower dependent smokers from more disadvantaged backgrounds aged 18-20, who have less motivation to quit.

Two-Photon Nanolithography of Tailored Hollow three-dimensional Microdevices for Biosystems.

Functional three-dimensional (3D) microstructures incorporating accessible interiors have emerged as a versatile platform for biosystem applications. By configuring their 3D geometric features, these biosystem microdevices can accurately evaluate and control targeted bioenvironments. However, classical fabrication techniques based on photolithography-etching processes cannot precisely and programmably control the geometric of the entire hollow 3D microstructures. Here, we proposed the use of a two-photon polymerization (TPP)-based technique for the precise, straightforward, and customizable preparation of hollow 3D microstructure devices with small opening(s). Factors governing the formation of hollow 3D biosystem microdevices, including material composition, laser input, and (post-) development treatment, have been systematically investigated and a set of optimized conditions are presented as a starting point for the development of novel hollow biosystem microdevices. To evaluate the broad applicability of this approach, a series of tailored hollow 3D microdevices with small opening(s), including a micropore, microneedle, microelectrode, microvalve, and micromachine, were successfully prepared using our direct laser writing-TPP technique. To further validate the feasibility of these biosystem microdevices in practical implementations, we demonstrated the use of hollow 3D micropore devices for the robust resistive-pulse analysis of nanoparticles.

A high-resolution study of in situ surface-enhanced Raman scattering nanotag behavior in biological systems.

The colloidal stability of surface-enhanced Raman scattering (SERS) nanotags (Raman reporter-conjugated plasmonic nanoparticles) significantly affects the accuracy and reproducibility of SERS measurements, particularly in biological systems. Limited understanding of SERS nanotag stability may partly hamper the translation of SERS nanotags from the laboratory to their use in the clinic. In this contribution, we utilized differential centrifugal sedimentation (DCS), a reliable and straightforward technique to comprehensively analyze the colloidal stability of SERS nanotags in biological systems. Compared with other particle characterization techniques, DCS has been shown to have a unique advantage for high-resolution and high-throughput polydisperse particle characterization. DCS data revealed that the universal aggregation prevention practice of coating SERS nanotags with silica or bovine serum albumin layers did not sufficiently stabilize them in common measurement environments (e.g., 1 × PBS). Combined DCS and SERS measurements established a strong correlation between the degrees of nanotag aggregation and signal intensities, further reinforcing the necessity of characterizing SERS nanotag stability for every condition in which they are used. We also found that increasing the protein thickness by the inclusion of extra protein components in the detection environments and antibody functionalization can improve the stability of SERS nanotags. We believe that this study can provide guidelines on appropriate measurement techniques and particle design considerations to assess and improve SERS nanotag stability in complex biological systems.

Retooling phage display with electrohydrodynamic nanomixing and nanopore sequencing.

Phage display methodologies offer a versatile platform for the isolation of single-chain Fv (scFv) molecules which may be rebuilt into monoclonal antibodies. Herein, we report on a complete workflow termed PhageXpress, for rapid selection of single-chain Fv sequences by leveraging electrohydrodynamic-manipulation of a solution containing phage library particles to enhance target binding whilst minimizing non-specific interactions. Our PhageXpress technique is combined with Oxford Nanopore Technologies' MinION sequencer and custom bioinformatics to achieve high-throughput screening of phage libraries. We performed 4 rounds of biopanning against Dengue virus (DENV) non-structural protein 1 (NS1) using traditional methods (4 week turnaround), which resulted in the isolation of 19 unique scFv clones. We validated the feasibility and efficiency of the PhageXpress method utilizing the same phage library and antigen target. Notably, we successfully mapped 14 of the 19 anti-NS1 scFv sequences (∼74%) with our new method, despite using ∼30-fold less particles during screening and conducting only a single round of biopanning. We believe this approach supersedes traditional methods for the discovery of bio-recognition molecules such as antibodies by speeding up the process for the development of therapeutic and diagnostic biologics.

Maskless 3D Ablation of Precise Microhole Structures in Plastics Using Femtosecond Laser Pulses.

Femtosecond laser ablation is a robust tool for the fabrication of microhole structures. This technique has several advantages compared to other microfabrication strategies for reliably preparing microhole structures of high quality and low cost. However, few studies have explored the use of femtosecond laser ablation in plastic materials because of the lack of controllability over the fabrication process in plastics. In particular, the depth profile of microhole structures prepared by conventional laser ablation techniques in plastics cannot be precisely and reproducibly controlled. In this paper, a novel three-dimensional femtosecond laser ablation technique was developed for the rapid fabrication of precise microhole structures in multiple plastics in air. Using a three-step fabrication scheme, microholes demonstrated extremely clean and sharp geometric features. This new technique also enables the precise creation of arbitrary-shaped microwell structures in plastic substrates through a rapid single-step ablation process, without the need for any masks. As a proof of concept for practical applications, precise microhole structures prepared by this novel femtosecond laser ablation technique were exploited for robust resistive-pulse sensing of microparticles.

Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing.

Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of pure yields of exosomes from complex biological fluids whilst preserving their physical characteristics is critical for downstream applications. In this study, we use 100 nm-liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as a model system as a model system to assess the effect of exosome isolation protocols on vesicle recovery and size distribution using a single-particle analysis method. We demonstrate that liposome size distribution and ζ-potential are comparable to extracted exosomes, making them an ideal model for comparison studies. Four different purification protocols were evaluated, with liposomes robustly isolated by three of them. Recovered yields varied and liposome size distribution was unaltered during processing, suggesting that these protocols do not induce particle aggregation. This leads us to conclude that the size distribution profile and characteristics of vesicles are stably maintained during processing and purification, suggesting that reports detailing how exosomes derived from tumour cells differ in size to those from normal cells are reporting a real phenomenon. However, we hypothesize that larger particles present in most purified exosome samples represent co-purified contaminating non-exosome debris. These isolation techniques are therefore likely nonspecific and may co-isolate non-exosome material of similar physical properties.

Treatment of infantile hemangiomas with propranolol: clinical guidelines.

BACKGROUND: Infantile hemangioma is a vascular tumor and requires treatment in lesions manifested by potentially dangerous symptoms. Several publications have reported that involution of infantile hemangioma could be accelerated by propranolol but have used only invalidated subjective measures of assessment. The authors aimed to objectively validate the aesthetic results after propranolol treatment for infantile hemangioma, and to produce a therapy protocol, including optimal timing for introduction, pretreatment preparation, dosage, frequency of visits, duration, and patient safety. METHODS: For the nonrandomized comparative cohort study, the authors enrolled 60 patients treated with propranolol. Medical two-dimensional photographs, taken before and after treatment, were subjectively analyzed by three plastic surgery consultants and objectively analyzed with a computer program. Aesthetic results were analyzed using the following parameters: subjective overall outcome, subjective color fading, and objective color fading. Reliability of subjective and objective methods was quantified and compared, as described with accuracy and repeatability. Volumetric parameters were obtained from three-dimensional scans taken before and after treatment and objectively analyzed with a computer program. Numerous patients' data were recorded from the medical notes. RESULTS: This study proved high efficiency of propranolol in treatment of infantile hemangioma, as assessed with the objective measures for the first time. The authors outlined an optimal treatment protocol, including introduction, dosage, duration, and cessation of therapy. CONCLUSIONS: Propranolol is an effective, well-tolerated, and safe first-line treatment for proliferative hemangioma. Therapy should begin early, continue with the target dosage of 2 mg/kg/day in three divided doses through the proliferative phase of infantile hemangioma, and be stopped gradually. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Nanoparticle ζ-potential measurements using tunable resistive pulse sensing with variable pressure.

Modern resistive pulse sensing techniques can be used to measure nanoparticle electrophoretic mobility, and hence ζ-potential. In contrast to conventional light scattering methods, resistive pulse sensing produces particle-by-particle data. We have used tunable resistive pulse sensing (TRPS) to compare methods for measuring the ζ-potential of carboxylated polystyrene nanoparticles. The five particle sets studied had nominal surface charge density (σ) between 0 and -0.67 C m(-2), and diameters in the range 160-230 nm. Data were collected with pressure in the range ±500 Pa applied across a tunable pore. In each experiment, pressure was varied either continuously or in discrete steps. Calculations of the ζ-potential were obtained by analysing both the rate and the full-width half maximum duration of resistive pulses. Data obtained from duration analyses were more reproducible than rate methods, yielding typical variations smaller than ±5 mV. When σ was greater (less negative) than -0.32 C m(-2), all of the analysis methods studied yielded a monotonic relationship between ζ-potential and σ. Complicated pulse data were observed near the pressure at which the net particle flux is zero, and these observations have been explored by examining competition between electrokinetic and pressure-driven transport. The typical difference between ζ-potentials obtained using TRPS and phase analysis light scattering was 15% (<5 mV), with an experimental error of ∼10% attributable to both techniques.