Direct Quantification Research Articles

Detection of Exchangeable Protons in NMR Metabolomic Analysis Using AI-Designed Water Irradiation Devoid Pulses.

1H NMR spectroscopy has enabled the quantitative profiling of metabolites in various biofluids, emerging as a possible diagnostic tool for metabolic disorders and other diseases. To boost the signal-to-noise ratio and detect proton resonances near the water signal, current 1H NMR experiments require solvent suppression schemes (e.g., presaturation, jump-and-return, WATERGATE, excitation sculpting, etc.). Unfortunately, these techniques affect the quantitative assessment of analytes containing exchangeable protons. To address this issue, we introduce two new one-dimensional (1D) 1H NMR techniques that eliminate the water signal, preserving the intensities of exchangeable protons. Using GENETICS-AI, a software that combines an evolutionary algorithm and artificial intelligence, we tailored new water irradiation devoid (WADE) pulses and optimized the 1D 1H NOESY sequence for metabolomic analysis. When applied to human urine samples, kidney tissue extract, and plasma, the WADE technique allowed for accurate measurement of typical metabolites and direct quantification of urea, which is usually challenging to measure using standard NMR experiments. We anticipate that these new NMR techniques will significantly improve the accuracy and reliability of metabolite quantitative assessment for a wide range of biological fluids.

Engineering Protein-Based Lipid-Binding Nanovesicles via Catechol-Amine-Derived Coacervation with Their Underlying Interfacial Mechanisms.

The development of nonphospholipid nanovesicles has garnered tremendous attention as a viable alternative to traditional liposomal nanovesicles. Protein/peptide-based nanovesicles have demonstrated their potential to reduce immunogenicity while enhancing bioactivity. However, a fundamental understanding of how proteinaceous vesicles interact with lipids and cell membranes remains elusive. In this study, we engineered a series of protamine-based nonphospholipid nanovesicles by modulating intramolecular catechol-amine interactions. By grafting trihydroxybenzene (GA) and catechol (CA) groups onto the protamine (Prot), a salt-triggered coacervation was observed in an alkaline environment with the size of as-prepared vesicles ranging from 200 to 1200 nm. The bonding affinity to lipid interfaces followed the order of Prot-CA-Fe3+(25 μM) > Prot-CA-Fe3+(10 μM) > Prot-CA > original Prot with the underlying nanomechanics investigated by the lipid bubble force measurement. Direct quantification of interactions between the nanovesicles and living human gingival fibroblasts was performed by using surface charge difference mapping. Introducing trace amounts of Fe3+ (at 10 and 25 μM) enhanced vesicle-lipid interactions via the synergy of catechol-amine interactions and Fe3+-induced complexation. This work provides improved valuable insights into the interactions between nanovesicles and cell membranes, offering an energetic paradigm for modulating cell-target delivery processes via intramolecular short-range interactions.

Biosensors Characterisation: Formal methods from the Perspective of Proteome Fractions

Abstract Many studies characterise transcription factors and other regulatory elements to control gene expression in recombinant systems. However, most lack a formal approach to analyse the inherent and context-specific variations of these regulatory components. This study addresses this gap by establishing a formal framework from which convenient methods are inferred to characterise regulatory circuits. We modelled the bacterial cell as a collection of proteome fractions. Deriving the time-dependent proteome fraction, we obtained a general theorem that describes its change as a function of its expression fraction, a specific portion of the total biosynthesis flux of the cell. Formal deduction reveals that when the proteome fraction reaches a maximum, it becomes equivalent to its expression fraction. This equation enables the reliable measurement of the expression fraction through direct protein quantification. In addition, the experimental data demonstrate a linear correlation between protein production rate and specific growth rate over a significant time period. This suggests a constant expression fraction within this window. For an IPTG biosensor, in five cellular contexts, expression fractions determined by the maximum method and the slope method produced strikingly similar dose-response parameters when independently fit to a Hill function. Furthermore, by analysing two more biosensors, for mercury and cumate detection, we demonstrate that the slope method can be applied effectively to various systems. Therefore, the concepts presented here provide convenient methods for obtaining dose-response parameters, clearly defining the time interval of their validity and offering a framework for interpreting typical biosensor outputs in terms of bacterial physiology.

A novel ddPCR™ assay for eDNA detection and quantification of Greater Amberjack Seriola dumerilli and three congeners in US waters: challenges and application to fisheries independent surveys.

Four Seriola species support recreational and commercial fisheries along the U.S. Atlantic Ocean and the Gulf of Mexico, with the S. dumerili Gulf of Mexico stock being overfished for over three decades. The study presented here is part of a fisheries-independent project initiated to determine an absolute abundance of S. dumerili, to expand biological knowledge of the species and to develop novel tools for fisheries management. Environmental DNA (eDNA) tools aimed at the detection and quantification of target species are starting to emerge in support of marine fisheries surveys. Key to progressing the field is Droplet Digital™ PCR (ddPCR™), a highly sensitive technique with advanced multiplexing and direct quantification capabilities that can provide fisheries scientists with improved interpretation of eDNA data. We developed and validated a novel tetraplex ddPCR™ assay able to detect and distinguish between S. dumerili, S. fasciata, S. rivoliana, and S. zonata from seawater eDNA samples. In order to groundtruth ddPCR™ data, and explore its capacity to provide abundance estimates, we compared ddPCR™ detections and quantifications to abundance data inferred from multiple camera (ROV, S-BRUV, chevron trap) and acoustic (VPS array) gears deployed during a fisheries research gear-calibration cruise. We demonstrated that with eDNA contamination controls and best practice protocols, it is viable to conduct eDNA research as part of a fisheries survey cruise. eDNA sampling was completed in less time than camera gears (15 min vs 2 h). Both eDNA and camera gears detected the presence of S. dumerili and S. rivoliana at both sites and all sampling days, but not S. fasciata and S. zonata. eDNA concentration data was higher for S. dumerili than S. rivoliana at both sites for all sampling days, in line with abundance patterns obtained from camera gears. The highest correlation (r = 0.97) was obtained between the measures of eDNA between gear deployments and ROV. Incorporating eDNA in fisheries surveys would not require additional days at sea and could improve precision in fish detection and abundance. eDNA can be a valuable complement to camera gears deployed in geographic areas or seasons with poor visibility conditions, where fish may be present but cannot be confidently identified to the species level. The high correlation obtained between ROV and eDNA data collected between gear deployments adds to a growing number of studies demonstrating the potential of eDNA as an indicator of abundance for fisheries stock assessments. Time-series data from a carefully designed eDNA survey, that estimates relative abundance, could be used as an index of relative abundance for the S. dumerili stock assessment. To achieve this, investment into follow-up studies with increased sample sizes and spatial and temporal replication would be necessary to allow for year-to-year comparisons and validate the robustness of the correlation observed.

Impact of measurement location on direct mitral regurgitation quantification using 4D flow CMR.

Four-dimensional (4D) flow cardiovascular magnetic resonance (CMR) shows promise for quantifying mitral regurgitation (MR) by allowing for direct regurgitant volume (RVol) measurement using a plane precisely placed at the MR jet. However, the ideal location of a measurement plane remains unclear. This study aims to systematically examine how varying measurement locations affect RVol quantification and determine the optimal location using the momentum conservation principle of a free jet. Patients diagnosed with MR by transthoracic echocardiography (TTE) and scheduled for CMR were prospectively recruited. Regurgitant jet flow volume (RVoljet) and regurgitant jet flow momentum (RMomjet) were quantified using 4D flow CMR at 7 locations along the jet axis, x. The reference plane (mid-plane, x=0mm) was positioned at the peak velocity of the jet at each cardiac phase, and 3 additional planes were positioned on either side of the jet, each 2.5mm apart. RVoljet was compared to RVolTTE, measured by the proximal isovelocity surface area method and RVolindirect, measured by subtracting aortic forward flow volume from the left ventricle stroke volume derived from 2D phase-contrast at the aortic valve and a stack of short-axis cine CMR techniques. RVoljet and RMomjet were quantified in 45 patients (age 63±13, male 26). In patients with RVoljet at x=0mm ≥ 10ml (n=25), RVoljet consistently increased as the plane moved downstream. RVoljet measured furthest upstream (x=-7.5mm) was significantly lower (39±11%, p<0.001) and RVoljet measured furthest downstream (x=7.5mm) was significantly higher (16±19%, p<0.001) than RVoljet at x=0mm. RMomjet similarly increased from x=-7.5 to 0mm (57±12%, p<0.001) but stabilized from x=0 to 7.5mm (-2±17%). From x=-7.5 to 7.5mm, RVoljet was in consistent moderate agreement with RVolindirect (n=41, bias=-2±24 to 8±32ml, ICC=0.55 to 0.63, p<0.001). The location of a measurement plane significantly influences RVol quantification using the direct 4D flow CMR approach. Based on the converging profile of RMomjet, we propose the peak velocity of the jet as the optimal position.

In-situ profiling of glycosylation on single cells with surface plasmon resonance imaging

Cellular glycosylation is crucial for cell recognition, signal transduction, and the development of various diseases, especially in tumor initiation, progression, and metastasis. Current glycosylation profiling methods normally involve laborious sample processing and labeling and lack in-situ quantitative analysis. Here, we present a direct optical method to investigate and quantify the glycan expression on single cells based on lectin-glycan kinetic quantification with plasmonic imaging. Three unlabeled lectins (WGA, SBA, ConA) are employed as probes to bind with specific glycans, and binding kinetics are assessed to determine glycosylation profiles. The result reveals cell-to-cell heterogeneity in glycosylation patterns. To demonstrate the capability of our method, the glycosylation profiling of four distinct cell lines is explored, showing obvious alterations in glycan expression related to tumor initiation, progression, and metastasis. This approach enables direct quantification of glycosylation and binding kinetics, providing insights into tumor cell glycosylation mechanisms and potential applications in disease diagnosis and treatment.

Focal Molography Allows for Affinity and Concentration Measurements of Proteins in Complex Matrices with High Accuracy

Characterizing biomolecular receptor–ligand interactions is critical for research and development. However, performing analyses in complex, biologically relevant matrices, such as serum, remains challenging due to non-specific binding that often impairs measurements. Here, we evaluated Focal Molography (FM) for determining KD and kinetic constants in comparison to gold-standard methods using single-domain heavy-chain antibodies in various systems. FM provided kinetic constants highly comparable to SPR and BLI in standard buffers containing blocking proteins, with KDs of soluble CD4 (sCD4) interactions within a 2.4-fold range across technologies. In buffers lacking blocking proteins, FM demonstrated greater robustness against non-specific binding and rebinding effects. In serum, FM exhibited stable baseline signals, unlike SPR and BLI, and yielded KDs of sCD4 interaction in 50% Bovine Serum within a 1.8-fold range of those obtained in standard buffers. For challenging molecules prone to non-specific binding (Granzyme B), FM successfully determined kinetic constants without external referencing. Finally, FM enabled direct analyte quantification in complex matrices. sCD4 quantification in cell culture media and 50% FBS showed recovery rates of 97.8–100.3% with an inter-assay CV below 1.3%. This study demonstrates the high potential of FM for kinetic affinity determination and biomarker quantification in complex matrices, enabling reliable measurements under biologically relevant conditions.

Aboveground-belowground linkages across vegetation degradation gradients differ among native eucalypt communities.

Native vegetation degradation impacts soil communities and their functions. However, these impacts are often studied by comparing soil biotic attributes across qualitatively defined, discrete degradation levels within a single plant community at a specific location. Direct quantification of the relationships between vegetation and soil attributes across continuous degradation gradients and at larger scales is rare but holds greater potential to reveal robust patterns in aboveground-belowground linkages that may apply across different plant communities. We investigated how native vegetation attributes relate to soil communities and their functions across a degradation gradient within three native temperate eucalypt woodland and forest communities that differed in soil nutrient availabilities. Across remnant patches of native vegetation in the Sydney Basin bioregion, we established plots representing different levels of decline in their vegetation quality (i.e., increased exotics and canopy changes) compared to relevant reference communities. In those plots, we assessed soil community groups (microbes and fauna), carbon (C) and nutrient cycling (litter decomposition, enzyme activity, and phosphate and nitrate accumulation rates), soil pH, texture and vegetation attributes (composition, structure, and function). Our unique study design revealed that the relationships between vegetation degradation and soil biota across the food web (i.e., AM fungi, Fungi:bacteria ratio, Gram-positive bacteria, total nematodes) were highly dependent on the plant community. However, the degradation impacts on soil functions (i.e., total enzyme activity, and phosphate availability) were mostly consistent, suggesting their potential as belowground indicators of ecosystem degradation, with a notable positive association observed in phosphate availability rates. Additionally, the effects of vegetation degradation on soil biota and their functions appeared stronger in the nutrient-poor plant communities, suggesting greater vulnerability of their belowground components. Our findings call for caution when generalizing belowground responses to degradation and for further research on how nutrient availability mediates the impacts of degradation on aboveground-belowground linkages.

Comparative Analysis of Viral Load Quantification Using Reverse Transcription Polymerase Chain Reaction and Digital Droplet Polymerase Chain Reaction.

In the year 2019, a highly virulent coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, precipitating the outbreak of the illness known as coronavirus disease 2019 (COVID-19). The commonly employed reverse transcription polymerase chain reaction (RT-qPCR) methodology serves to estimate the viral load in each patient's sample by employing a standard curve. However, it is imperative to recognize that this technique exhibits limitations with respect to clinical diagnosis and therapeutic applications, since an advancement of the conventional polymerase chain reaction methods, digital polymerase chain reaction (digital PCR or DDPCR), enables the direct quantification and clonal amplification of nucleic acid strands. The primary divergence between dPCR and traditional PCR resides in their approaches to measuring nucleic acid quantities. In this study, we investigated the viral loads of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) within 461 participants. By employing both RT-qPCR and DDPCR techniques, we established a comparison between the quantification methodologies of the two approaches. Our findings illustrate that the quantification through DDPCR affords a superior means of monitoring viral load within lower respiratory tract samples, thus enhancing the assessment of disease progression, particularly in scenarios characterized by low viral loads.

In situ quantification of ribosome number by electron tomography.

Ribosomes, discovered in 1955 by George Palade, were initially described as small cytoplasmic particles preferentially associated with the endoplasmic reticulum (ER). Over the years, extensive research has focused on both the structure and function of ribosomes. However, a fundamental question - how many ribosomes are present within whole cells - has remained largely unaddressed. In this study, we developed a microscopic method to quantify the total number of ribosomes in hTERT-RPE-1 cells and in nematode cells from various tissues of Caenorhabditis elegans hermaphrodites. Using electron tomography of high-pressure frozen, freeze-substituted and resin-embedded samples, we determined that the ribosome number in hTERT-RPE-1 cells is in the same order of magnitude as biochemical measurements obtained via RNA capillary electrophoresis. As expected, control worms exhibited a higher number of ribosomes compared to RNA polymerase I A subunit (RPOA-1)-depleted worms in two out of three analysed tissue types. Our imaging-based approach complements established biochemical methods by enabling direct quantification of ribosome numbers in specific samples. This method offers a powerful tool for advancing our understanding of ribosome localisation and distribution in cells and tissues across diverse model systems.

Direct high-precision radon quantification for interpreting high-frequency greenhouse gas measurements

Abstract. We present a protocol to improve confidence in reported radon activity concentrations, facilitating direct site-to-site comparisons and integration with co-located greenhouse gas (GHG) measurements within a network of three independently managed observatories in the UK. Translating spot measurements of atmospheric GHG amount fractions into regional flux estimates (“top-down” analysis) is usually performed with atmospheric transport models (ATMs), which calculate the sensitivity of regional emissions to changes in observed GHGs at a finite number of locations. However, the uncertainty of regional emissions is closely linked to ATM uncertainties. Radon, emitted naturally from the land surface, can be used as a tracer of atmospheric transport and mixing to independently evaluate the performance of such models. To accomplish this, the radon measurements need to have a comparable precision to the GHGs at the modelled temporal resolution. Australian Nuclear Science and Technology Organisation (ANSTO) dual-flow-loop two-filter radon detectors provide output every 30 min. The measurement accuracy at this temporal resolution depends on the characterization and removal of instrumental background, the calibration procedure, and response time correction. Consequently, unless these steps are standardized, measurement precision may differ between sites. Here we describe standardized approaches regarding (1) instrument maintenance, (2) quality control of the raw data stream, (3) determination and removal of the instrumental background, (4) calibration methods, and (5) response time correction (by deconvolution). Furthermore, we assign uncertainties for each reported 30 min radon estimate (assuming these steps have been followed) and validate the final result through comparison of diurnal and sub-diurnal radon characteristics with co-located GHG measurements. While derived for a network of UK observatories, the proposed standardized protocol could be equally applied to two-filter dual-flow-loop radon observations across larger networks, such as the Integrated Carbon Observation System (ICOS) or the Global Atmosphere Watch (GAW) baseline network.

Correction to “Novel Conductive and Redox-Active Molecularly Imprinted Polymer for Direct Quantification of Perfluorooctanoic Acid”

Correction to “Novel Conductive and Redox-Active Molecularly Imprinted Polymer for Direct Quantification of Perfluorooctanoic Acid”

Microfluidic Integration of Magnetically Functionalized Microwires for Flow Cytometry Protein Quantification.

A novel approach to protein quantification utilizing a microfluidic platform activated by a magnetic assembly of functionalized magnetic beads around soft magnetic capture centers is presented. Functionalized magnetic beads, known for their high surface area and facile manipulation under external magnetic fields, are injected inside microfluidic channels and immobilized magnetically on the surface of glass-coated soft magnetic microwires placed along the symmetry axis of these channels. A fluorescent (Cy5) immunomagnetic sandwich ELISA is then performed by sequentially flowing the sample and all necessary reagents in the microfluidic channels. Direct protein quantification is performed by magnetically releasing the beads from the microwire and evaluating their fluorescence intensity with the help of a miniature (microfluidic-based) flow cytometer. Measurements of ICAM-1 protein concentration in human blood plasma samples confirm the feasibility of the approach through extensive performance benchmarking. The automation and multiplexing capabilities of the proposed platform further demonstrate its potential for protein quantification in point-of-care settings using microfluidics and miniature flow cytometry instruments.

Coronary cameral fistula: Diagnosis and direct shunt quantification by cardiac MRI

Coronary cameral fistula: Diagnosis and direct shunt quantification by cardiac MRI

Vascularized tumor on a microfluidic chip to study mechanisms promoting tumor neovascularization and vascular targeted therapies.

The cascade of events leading to tumor formation includes induction of a tumor supporting neovasculature, as a primary hallmark of cancer. Developing vasculature is difficult to evaluate in vivo but can be captured using microfluidic chip technology and patient derived cells. Herein, we established an on chip approach to investigate the mechanisms promoting tumor vascularization and vascular targeted therapies via co-culture of cancer spheroids and endothelial cells in a three dimensional environment. Methods: We investigated both, tumor neovascularization and therapy, via co-culture of human derived endothelial cells and adjacently localized metastatic renal cell carcinoma spheroids on a commercially available microfluidic chip system. Metastatic renal cell carcinoma spheroids adjacent to primary vessels model tumor, and induce vessels to sprout neovasculature towards the tumor. We monitored real time changes in vessel formation, probed the interactions of tumor and endothelial cells, and evaluated the role of important effectors in tumor vasculature. In addition to wild type endothelial cells, we evaluated endothelial cells that overexpress Prostate Specific Membrane Antigen (PSMA), that has emerged as a marker of tumor associated neovasculature. We characterized the process of neovascularization on the microfluidic chip stimulated by enhanced culture medium and the investigated metastatic renal cell carcinomas, and assessed endothelial cells responses to vascular targeted therapy with bevacizumab via confocal microscopy imaging. To emphasize the potential clinical relevance of metastatic renal cell carcinomas on chip, we compared therapy with bevacizumab on chip with an in vivo model of the same tumor. Results: Our model permitted real-time, high-resolution observation and assessment of tumor-induced angiogenesis, where endothelial cells sprouted towards the tumor and mimicked a vascular network. Bevacizumab, an antiangiogenic agent, disrupted interactions between vessels and tumors, destroying the vascular network. The on chip approach enabled assessment of endothelial cell biology, vessel's functionality, drug delivery, and molecular expression of PSMA. Conclusion: Observations in the vascularized tumor on chip permitted direct and conclusive quantification of vascular targeted therapies in weeks as opposed to months in a comparable animal model, and bridged the gap between in vitro and in vivo models.

Direct quantification of the plasmon dephasing time in ensembles of gold nanorods through two-dimensional electronic spectroscopy.

In this study, we used two-dimensional electronic spectroscopy to examine the early femtosecond dynamics of suspensions of colloidal gold nanorods with different aspect ratios. In all samples, the signal distribution in the 2D maps at this timescale shows a distinctive dispersive behavior, which can be explained by the interference between the exciting field and the field produced on the nanoparticle's surface by the collective motion of electrons when the plasmon is excited. Studying this interference effect, which is active only until the plasmon has been dephased, allows for a direct estimation of the dephasing time of the plasmon of an ensemble of colloidal particles. Our findings provide insight into the fundamental behavior of plasmonic states and highlight the potential of two-dimensional electronic spectroscopy in uncovering ultrafast and coherent optical phenomena at the nanoscale.

Advanced imaging techniques in crystal arthritis.

Gout and calcium pyrophosphate deposition (CPPD) disease are the most common causes of crystal arthritis. Identifying the pathogenic crystal deposition is the cornerstone of the diagnosis, but also prognosis and monitoring of the diseases. Conventional radiography has been for decades the only imaging technique used, with its very restricted sensitivity in both diseases. Advanced techniques, namely ultrasound and dual-energy computed tomography (DECT), are being increasingly used in the diagnosis and management of gout and CPPD diseases, and their role is now well recognized in classification criteria and in recommendations for the diagnosis and management. In gout, ultrasound elementary lesions of monosodium urate deposition are well defined and have been shown to be sensitive to change and can be monitored, while direct quantification of these deposits can be performed with DECT. In CPPD disease, the definition of elementary lesions and their scoring has been well established for ultrasound, while the proof of concept that DECT can help discriminate calcium pyrophosphate crystal deposits among other calcium-containing structures has been shown. The aim of this narrative review is to provide an overview of the use of advanced imaging techniques in crystal-induced arthropathies.

A Streamlined High-Throughput LC-MS Assay for Quantifying Peptide Degradation in Cell Culture.

Peptides are widely used in biomaterials due to their ease of synthesis, ability to signal cells, and modify the properties of biomaterials. A key benefit of using peptides is that they are natural substrates for cell-secreted enzymes, which creates the possibility of utilizing cell-secreted enzymes for tuning cell-material interactions. However, these enzymes can also induce unwanted degradation of bioactive peptides in biomaterials, or in peptide therapies. Liquid chromatography-mass spectrometry (LC-MS) is a widely used, powerful methodology that can separate complex mixtures of molecules and quantify numerous analytes within a single run. There are several challenges in using LC-MS for the multiplexed quantification of cell-induced peptide degradation, including the need for nondegradable internal standards and the identification of optimal sample storage conditions. Another problem is that cell culture media and biological samples typically contain both proteins and lipids that can accumulate on chromatography columns and degrade their performance. Removing these constituents can be expensive, time-consuming, and increases sample variability. However, loading unpurified samples onto the column without removing lipids and proteins will foul the column. Here, we show that directly injecting complex, unpurified samples onto the LC-MS without any purification enables rapid and accurate quantification of peptide concentration and that hundreds of LC-MS runs can be done on a single column without significantly diminishing the ability to quantify the degradation of peptide libraries. To understand how repeated injections degrade column performance, a model library was injected into the LC-MS hundreds of times. It was then determined that column failure is evident when hydrophilic peptides are no longer retained on the column and that failure can be easily identified by using standard peptide mixtures for column benchmarking. In total, this work introduces a simple and effective method for simultaneously quantifying the degradation of dozens of peptides in cell culture. By providing a streamlined and cost-effective method for the direct quantification of peptide degradation in complex biological samples, this work enables more efficient assessment of peptide stability and functionality, facilitating the development of advanced biomaterials and peptide-based therapies.

Improved Voltammetric Procedure for Chloride Determination in Highly Acidic Media.

Cyclic voltammetry (CV) can be applied as a reliable method for the determination of chloride ions in a range from several to a couple hundred (about 200) ppm. Since the standard potential of chloride ion/gaseous chlorine is 1.36 V vs. normal hydrogen electrode (NHE), the efficient oxidation of Cl- ion occurs at very positive electrode potentials, usually higher than +1.7 V vs. NHE. It is possible to observe this phenomenon only at noble-metal or inert carbon electrodes. Many other solutes, usually organic compounds, are often oxidized at this potential. This is the reason why the determination of Cl- content directly from an increase in the oxidation current is not reliable and could lead to overestimated values. However, gaseous chlorine, generated at a positive potential dissolve in the analyzed solution, could be reduced in the reverse scan of a cyclic voltammetric curve. Optimization of the experimental procedure using statistical tools enables the development of an improved method for the direct quantification of chloride ions in acid copper electroplating baths. Under the proposed experimental conditions, the calibration curve (Cl2 voltammetric reduction current vs. chloride ions concentration) is represented by the linear model for the concentration of chlorides ranging from 10 to 200 mg/dm3. The developed method for analyzing chloride ions in an acid sulfate electroplating copper bath has many unique properties. It is fast; the time of a single analysis is less than 20 min. In automatic mode, it can be repeated up to 50 times a day. The method does not require processing of the sample of the analyzed bath before measurement. As a result, no additional chemical reagents are used, and the test sample can be returned to the plating bath.

Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.

Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in translational studies for genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.