Expression of Interferon Epsilon in Mucosal Epithelium is Regulated by Elf3.

Interferon epsilon (IFNε) is a unique type I interferon (IFN) that shows distinct constitutive expression in reproductive tract epithelium. Understanding how IFNε expression is regulated is critical for the mechanism of action in protecting the mucosa from infection. Combined computational and experimental investigation of the promoter of IFNε predicted transcription factor binding sites for the ETS family of transcription factors. We demonstrate here that Ifnε is regulated by Elf3, an epithelial restricted member of the ETS family. It is co-expressed with IFNε at the epithelium of uterus, lung and intestine, and we focused on regulation of IFNε expression in the uterus. Promoter reporter studies demonstrated that Elf3 was a strong driver of Ifnε expression; knockdown of Elf3 reduced expression levels of IFNε; Elf3 regulated Ifnε expression and chromatin immunoprecipitation (ChIP) confirmed the direct binding of Elf3 to the IFNε promoter. These data show that Elf3 is important in regulating protective mucosal immunity by driving constitutive expression of IFNε to protect mucosal tissues from infection in at least three organ systems.

The utilization of open-source microcontroller boards and single-board computers in liquid chromatography.

Electronic system control of analytical instrumentation remains a critical aspect of modern measurement science. Within the field of liquid chromatography (LC), this is especially relevant for automation, module operation, detection, data acquisition, and data analysis. Increasingly, home-built analytical tools used for liquid-phase separations rely upon open-source microcontrollers and single-board computers to aid in simplifying these operations. In this review, we detail literature reported within the past 5 years in which these types of devices were used to advance various aspects of the LC research field, including sample preparation, instrument control, and data collection.

High-Throughput Capillary Liquid Chromatography Using a Droplet Injection and Application to Reaction Screening.

The cycle time of a standard liquid chromatography (LC) system is the sum of the time for the chromatographic run and the autosampler injection sequence. Although LC separation times in the 1-10 s range have been demonstrated, injection sequences are commonly >15 s, limiting throughput possible with LC separations. Further, such separations are performed on relatively large bore columns requiring flow rates of ≥5 mL/min, thus generating large volumes of mobile phase waste when used for large scale screening and increasing the difficulty in interfacing to mass spectrometry. Here, a droplet injector system was established that replaces the autosampler with a four-port, two-position valve equipped with a 20 nL internal loop interfaced to a syringe pump and a three-axis positioner to withdraw sample droplets from a well plate. In the system, sample and immiscible fluid are pulled alternately from a well plate into a capillary and then through the injection valve. The valve is actuated when sample fills the loop to allow sequential injection of samples at high throughput. Capillary LC columns with 300 µm inner diameter were used to reduce the consumption of mobile phase and sample. The system achieved 96 separations of 20 nL droplet samples containing 3 components in as little as 8.1 min with 5-s cycle time. This system was coupled to a mass spectrometer through an electrospray ionization source for high-throughput chemical reaction screening.

Patient-Derived Organoids as Therapy Screening Platforms in Cancer Patients.

Patient-derived organoids (PDOs) developed ex vivo and in vitro are increasingly used for therapeutic screening. They provide a more physiologically relevant model for drug discovery and development compared to traditional cell lines. However, several challenges remain to be addressed to fully realize the potential of PDOs in therapeutic screening. This paper summarizes recent advancements in PDO development and the enhancement of PDO culture models. This is achieved by leveraging materials engineering and microfabrication technologies, including organs-on-a-chip and droplet microfluidics. Additionally, this work discusses the application of PDOs in therapy screening to meet diverse requirements and overcome bottlenecks in cancer treatment. Furthermore, this work introduces tools for data processing and analysis of organoids, along with their microenvironment. These tools aim to achieve enhanced readouts. Finally, this work explores the challenges and future perspectives of using PDOs in drug development and personalized screening for cancer patients.

Exploring Biopharmaceutical Analysis with Compact Capillary Liquid Chromatography Instrumentation.

A recent trend in the design of liquid chromatography (LC) instrumentation is the move towards miniaturized and portable systems. These smaller platforms provide wider flexibility in operation, with the opportunity for conducting analysis directly at the point of sample collection rather than transporting the sample to a centralized laboratory facility. For the manufacturing of pharmaceutical and biopharmaceutical products, these platforms can be implemented for process monitoring and product characterization directly in manufacturing environments. This article describes a portable, miniaturized LC instrument coupled to a mass spectrometer (MS) for characterization of a biopharmaceutical monoclonal antibody (mAb).

Column selection considerations in compact capillary liquid chromatography.

Recent years have seen significant advances in compact, portable capillary LC instrumentation. This study explores the performances of several commercially available columns within the pressure and flow limits of both the columns and one of these compact LC instruments. The commercially available compact capillary LC system with UV-absorbance detector used in this study is typically operated using columns in the 0.15-0.3 mm internal diameter (i.d.) range. Efficiency measurements (i.e., theoretical plates, N) for six columns with i.d.s in this range and of varying lengths and pressure limits, packed with stationary phases of different particle diameters and morphologies, were made using a mixture of standard alkylphenones. Kinetic plot comparisons between columns that vary by one (or more) of these parameters are described, along with calculated kinetic performance and Knox-Saleem limits. These theoretical performance descriptions provide insight into optimal operating conditions when using capillary LC systems. Based on kinetic plot evaluation of available capillary columns in the 0.2-0.3 mm i.d. range with a conservative upper pressure limit of 330 bar packed with superficially porous particles, a 25 cm column could generate ∼47,000 plates in 7.85 min when operated at 2.4 µL/min. For comparison, more robust 0.3 mm i.d. columns (packed with fully porous particles) that can be operated at higher pressures than can be provided by the pumping system (conservative pump upper pressure limit of 570 bar), a ∼20 cm column could generate nearly 40,000 plates in 5.9 min if operated at 6 µL/min. Across all capillary LC columns measured, higher pressure limits and shorter columns can provide the best throughput when considering both speed and efficiency.

Development of a dual-electrospray ionization source with in-line absorbance-based voltage control.

Emitter tip arrays for electrospray ionization have been used for a variety of MS sample introduction purposes, including detection of multiple sample eluent streams and improved accuracy through parallel infusion of an internal standard. User control is typically required for targeted application of high voltage to specific channels to maximize analyte signal and minimize other background signals. In this communication, an automated approach to applying electrospray voltage only when a detectable analyte is present is described. An in-line absorbance detector is used to identify the presence of an analyte in the fluidic path between the sample introduction valve and the mass spectrometer. A Raspberry Pi-controlled system is then used to apply high voltage to a downstream emitter tip at the MS inlet following a delay volume between the detectors. Demonstration of this technique on two parallel sample channels is reported, including a pulsed voltage application to maximize signal when analytes elute on each channel simultaneously.

A review of two-dimensional liquid chromatography approaches using parallel column arrays in the second dimension.

Multi-dimensional liquid chromatography techniques play an important role in the analysis of complex mixtures. The keys to maximizing peak capacity in these methods are fast sampling rates and sufficient complementarity between the first- (1D) and second- (2D) dimension separations. One way that these criteria have been met is by using 2D parallel column arrays. This review covers demonstrations of this approach in the literature that have been published over the past three decades. Two or more identical 2D columns can be operated in a sequential order to permit increased separation times and higher peak capacities in the second dimension without the concomitant decrease in sampling rate. The parallel column arrays can also be operated simultaneously to reduce total analysis time. Columns with different stationary phase chemistries can be used in the 2D column array to increase complementarity by utilizing specific stationary phases for various first dimension fractions. More recently, this type of platform has been used to automate the development of two-dimensional (2D) achiral-chiral LC methods. These strategies, as well as recent efforts toward the development of integrated, spatial multi-dimensional LC devices that include parallel column arrays, are discussed here.

Implementing 1.5 mm internal diameter columns into analytical workflows.

The use of smaller column diameters in liquid chromatography (LC) is often associated with capillary LC. Although there are many analytical benefits gained by adapting this format, routine use continues to be challenging due to column fragility and extra column dispersion. Bridging the gap between routinely used 2.1 mm columns and capillary bore columns allows for a sequential but far from insignificant increase in performance without the need for specialized equipment associated with very low dispersion LC systems. Moreover, an incremental decrease in column internal diameter (i.d.) allows for similar mass load (avoiding column overload that may be observed in much larger decreases in i.d. without trapping) and thus an increase in measured signal. As such, 1.5 mm i.d. columns provide an alternative intermediate dimension between the more regularly used 2.1 mm i.d. columns and 1 mm i.d. columns. These columns balance an increase in sensitivity compared to 2.1 mm i.d. columns (theoretically doubling the time-domain peak area in mass sensitive detectors for the same mass load), while mitigating the efficiency losses due to extra-column dispersion effects that are commonly observed with 1.0 mm i.d. columns. Here, the use of 1.5 mm i.d. columns was applied to LC/UV analysis of small molecules and LC/MS methods for the analysis of monoclonal antibodies. With equivalent mass load on column, the 1.5 mm i.d. columns provide two-to-threefold improvement in analyte peak area signal for small molecules as well as intact, subunit, and peptide levels of antibody analysis. Peak height was also increased using the 1.5 mm i.d. column, although the scale of increase varies between isocratic and gradient modes, likely due to differences in system dispersion effects and variation in electrospray ionization efficiency at different flow rates.

Online Monitoring of Small Volume Reactions Using Compact Liquid Chromatography Instrumentation.

A wide variety of analytical techniques have been employed for monitoring chemical reactions, with online instrumentation providing additional benefits compared to offline analysis. A challenge in the past for online monitoring has been placement of the monitoring instrumentation as close as possible to the reaction vessel to maximize sampling temporal resolution and preserve sample composition integrity. Furthermore, the ability to sample very small volumes from bench-scale reactions allows the use of small reaction vessels and conservation of expensive reagents. In this study, a compact capillary LC instrument was used for online monitoring of as small as 1 mL total volume of a chemical reaction mixture, with automated sampling of nL-scale volumes directly from the reaction vessel used for analysis. Analyses to demonstrate short term (~2 h) and long term (~ 50 h) reactions were conducted using tandem on-capillary ultraviolet absorbance followed by in-line MS detection or ultraviolet absorbance detection alone, respectively. For both short term and long term reactions (10 and 250 injections, respectively), sampling approaches using syringe pumps minimized the overall sample loss to ~0.2% of the total reaction volume.

Measurement of optimal flow rate in gradient elution liquid chromatography.

Method development in gradient LC relies upon the selection of a solvent time program and a mobile phase flow rate. The flow rate, optimal for gradient separation cannot be inherently predicted by the isocratic value optimal for a given analyte, and rather should be identified independently to ensure the highest separation performance of gradient analysis. The optimal flow rate (Fopt) is defined herein as the solvent volumetric flow rate (F) maximizing the separation (Δs) of a predetermined peak-pair or the separation capacity (sc) of the entire LC analysis. The theoretical background and the experimental technique of measurement of Fopt in gradient elution analysis were considered and experimentally demonstrated. The technique of measuring Fopt is based on translatable changes of F where the product FtG (tG is the gradient time) was the same for all values of F. The Fopt was found as F corresponding to the maximum in Δs or in sc.

Comparison of Design Approaches for Low-Cost Sampling Mechanisms in Open-Source Chemical Instrumentation.

Robotic positioning systems are used in a variety of chemical instruments, primarily for liquid handling purposes, such as autosamplers from vials or well plates. Here, two approaches to the design of open-source autosampler positioning systems for use with 96-well plates are described and compared. The first system, a 3-axis design similar to many low-cost 3D printers that are available on the market, is constructed using an aluminum design and stepper motors. The other system relies upon a series of 3D printed parts to achieve movement with a series of linker arms based on Selective Compliance Assembly Robot Arm (SCARA) design principles. Full printer design files, assembly instructions, software, and user directions are included for both samplers. The positioning precision of the 3-axis system is better than the SCARA mechanism due to finer motor control, albeit with a slightly higher cost of materials. Based on the improved precision of this approach, the 3-axis autosampler system was used to demonstrate the generation of a segmented flow droplet stream from adjacent wells within a 96-well plate.

Utility of low-cost, miniaturized peristaltic and Venturi pumps in droplet microfluidics.

Many laboratory applications utilizing droplet microfluidics rely on precision syringe pumps for flow generation. In this study, the use of an open-source peristaltic pump primarily composed of 3D printed parts and a low-cost commercial Venturi pump are explored for their use as an alternative to syringe pumps for droplet microfluidics. Both devices provided stable flow (<2% RSD) over a range of 1-7 µL/min and high reproducibility in signal intensity at a droplet generation rate around 0.25 Hz (<3% RSD), which are comparable in performance to similar measurements on standard syringe pumps. As a novel flow generation source for microfluidic applications, the use of the miniaturized Venturi pump was also applied to droplet signal monitoring studies used to measure changes in concentration over time, with average signal reproducibility <4% RSD for both single-stream fluorometric and reagent addition colorimetric applications. These low-cost flow methods provide stable flow sufficient for common droplet microfluidic approaches and can be implemented in a wide variety of simple, and potentially portable, analytical measurement devices.

Low-cost and open-source strategies for chemical separations.

In recent years, a trend toward utilizing open access resources for laboratory research has begun. Open-source design strategies for scientific hardware rely upon the use of widely available parts, especially those that can be directly printed using additive manufacturing techniques and electronic components that can be connected to low-cost microcontrollers. Open-source software eliminates the need for expensive commercial licenses and provides the opportunity to design programs for specific needs. In this review, the impact of the "open-source movement" within the field of chemical separations is described, primarily through a comprehensive look at research in this area over the past five years. Topics that are covered include general laboratory equipment, sample preparation techniques, separations-based analysis, detection strategies, electronic system control, and software for data processing. Remaining hurdles and possible opportunities for further adoption of open-source approaches in the context of these separations-related topics are also discussed.

Comparison of real-world treatment outcomes of femtosecond laser-assisted cataract surgery and phacoemulsification cataract surgery: A retrospective, observational study from an outpatient clinic in France.

PURPOSE: This study compared real-world safety and efficacy outcomes of cataract surgery performed with LenSx femtosecond laser-assisted cataract surgery or manual phacoemulsification cataract surgery procedures. METHODS: A retrospective observational study used data from anonymised electronic medical records to compare mean cumulative dissipated energy, the proportion of eyes reaching emmetropia, mean change in best-corrected distance visual acuity and the proportion of eyes with post-surgical complications, including corneal oedema and posterior capsule opacification. Results were adjusted for multiple comparisons for primary and secondary objectives. RESULTS: Data from 811 phacoemulsification cataract surgery and 496 femtosecond laser-assisted cataract surgery procedures were analysed. Mean cumulative dissipated energy was significantly lower for femtosecond laser-assisted cataract surgery (6.5 percent-seconds) than for phacoemulsification cataract surgery (14.3 percent-seconds; p < 0.0001) procedures. More femtosecond laser-assisted cataract surgery (81.2%) procedures achieved emmetropia (⩽ 0.5 dioptre) than did phacoemulsification cataract surgery (73.5%) procedures, although this difference was not statistically significant. Mean change in best-corrected distance visual acuity and the proportion of eyes with corneal oedema, posterior capsule opacification or other complications were not significantly different between cohorts when adjusted for multiple comparisons. CONCLUSIONS: In this single-centre, single-surgeon retrospective electronic medical record database study using divide and conquer technique, femtosecond laser-assisted cataract surgery was associated with significantly lower cumulative dissipated energy when compared to manual phacoemulsification cataract surgery. This supports the hypothesis that femtosecond laser-assisted cataract surgery involves less mechanical trauma, which might lead to more consistent refractive and safety outcomes than manual phacoemulsification cataract surgery, though such outcomes were found to be comparable in this study.

Portable capillary liquid chromatography for pharmaceutical and illicit drug analysis.

A newly developed portable capillary liquid chromatograph was investigated for the separation of various pharmaceutical and illicit drug compounds. The system consists of two high-pressure syringe pumps capable of delivering capillary-scale flow rates at pressures up to 10 000 psi. Capillary liquid chromatography columns packed with sub-2 µm particles are housed in cartridges that can be inserted into the system and easily connected through high-pressure fluidic contact points by simply applying a specific, predetermined torque rather than using standard fittings and less precise sealing protocols. Several over-the-counter analgesic drug separations are demonstrated, along with a simple online measurement of tablet dissolution. Twenty illicit drug compounds were also separated across six targeted drug panels. The results described in this study demonstrate the capability of this compact liquid chromatography instrument to address several important drug-related applications while simplifying system operation, and greatly reducing solvent usage and waste generation essential for onsite analysis.

A low-cost, open-source digital stripchart recorder for chromatographic detectors using a Raspberry Pi.

One of the most critical aspects of chromatographic analysis is effective data acquisition and processing. Typical approaches include software platforms designed for specific instruments or commercial data acquisition hardware boards, both of which require expensive licenses to use and operate. To increase the access and affordability of chromatographic data acquisition, especially for systems in which software control has become obsolete or must be written in-house, an open-source digital stripchart recorder has been developed. This system is built upon a Raspberry Pi single-board computer and a plug-in printed circuit board with the necessary integrated circuits for data acquisition. Using an open-source software called Processing, a complete user interface to control the system was developed that enables the acquisition, filtering, and processing of chromatographic data. The system performs comparably to more expensive platforms, with calculated values for peak area, retention time, and plate count all within 3% of the values calculated by a widely used commercial chromatography data software package.

Carrier motion in as-spun and annealed P3HT:PCBM blends revealed by ultrafast optical electric field probing and Monte Carlo simulations.

Charge transport dynamics in solar cell devices based on as-spun and annealed P3HT:PCBM films are compared using ultrafast time-resolved optical probing of the electric field by means of field-induced second harmonic generation. The results show that charge carriers drift about twice as far during the first 3 ns after photogeneration in a device where the active layer has been thermally annealed. The carrier dynamics were modelled using Monte-Carlo simulations and good agreement between experimental and simulated drift dynamics was obtained using identical model parameters for both cells, but with different average PCBM and polymer domain sizes. The calculations suggest that small domain sizes in as-spun samples limit the carrier separation distance disabling their escape from geminate recombination.