Single Step Research Articles

A Light-Powered Single-Stranded DNA Molecular Motor with Colour-Selective Single-Step Control.

Top-down control of small motion is possible through top-down controlled molecular motors in replacement of larger actuators like MEMS or NEMS (micro- or nano-electromechanical systems) in the current precision technology. Improving top-down control of molecular motors to every single step is desirable for this purpose, and also for synchronization of motor actions for amplified effects. Here we report a designed single-stranded DNA molecular motor powered by alternated ultraviolet and visible light for processive track-walking, with the two light colours each locking the motor in a full directional step to allow saturated driving but no overstepping. This novel nano-optomechanical driving mechanism pushes the top-down control of molecular motors down to every single step, thus providing a key technical capability to advance the molecular motor-based precision technology and also motor synchronization for amplified effects.

A Light‐Powered Single‐Stranded DNA Molecular Motor with Colour‐Selective Single‐Step Control

AbstractTop‐down control of small motion is possible through top‐down controlled molecular motors in replacement of larger actuators like MEMS or NEMS (micro‐ or nano‐electromechanical systems) in the current precision technology. Improving top‐down control of molecular motors to every single step is desirable for this purpose, and also for synchronization of motor actions for amplified effects. Here we report a designed single‐stranded DNA molecular motor powered by alternated ultraviolet and visible light for processive track‐walking, with the two light colours each locking the motor in a full directional step to allow saturated driving but no overstepping. This novel nano‐optomechanical driving mechanism pushes the top‐down control of molecular motors down to every single step, thus providing a key technical capability to advance the molecular motor‐based precision technology and also motor synchronization for amplified effects.

Interpenetrated In(III)-MOF with Multiple Recognition Sites for Single-Step Ethylene Purification.

An interpenetrated indium(III) metal-organic framework (MOF), NTUniv-73, with a rarely reported tetrameric indium cluster is developed for streamlining ethylene purification from C2 gases. At 298 K, the adsorption capacities exhibited a complete reversal sequence of C2H6 > C2H2 > C2H4. Grand canonical Monte Carlo simulation indicated that the corners in a octahedral cage facilitated the C2H2/C2H4 separation, while the pocket-like aperture situated between adjacent octahedral cages allows for full contact of C2H6. Breakthrough experiments illustrated that NTUniv-73 could yield pure C2H4 in a single step with a productivity of 0.42 mmol g-1.

PaIRS-UniNa: A Robust And Accurate Free Tool For Digital Particle Image Velocimetry And Optical Camera Calibration

This contribution presents PaIRS-UniNa (PArticle Image Reconstruction Software – University of Naples “Federico II”), a free software application designed to perform digital particle image velocimetry (PIV). PaIRS-UniNa relies on a powerful C library designed for speed, robustness and accuracy, and a user-friendly graphical interface (Figure 1) running in Python environment on all operating systems, including Windows, Linux and MacOS. At the current stage, it features modules to perform two-component (2C) PIV, stereoscopic PIV and optical camera calibration for multi-camera systems. The processing of PIV and stereoscopic PIV data is based on an iterative image deformation method, which can be customized by the user varying different parameters in such a way to find the best compromise between accuracy and computational speed. These parameters include the size, shape and number of interrogation windows, the number of process iterations and the type of interpolating functions for both image deformation and displacement interpolation (such as bilinear, bicubic, methods based on simplexes or the FFT shift theorem and B-splines of varying orders). Additionally, it is possible to select a range of weighting windows for cross-correlation estimation and velocity field filtering, including rectangular, Blackman, Gaussian, Harris, Hann windows, among others, and several vector validation strategies for outlier detection and replacement. Such a level of customization enables to effectively adjust the modulation transfer function of the PIV process to meet specific application requirements. For the stereoscopic PIV process two different approaches are possible: mapping, which involves applying PIV to de-warped images for 2D-2C displacement fields, and warping, which applies PIV to raw images and combine 3C reconstruction and image dewarping in a single subsequent step. In order to accurately estimate the laser sheet plane location and correct errors due to misalignment and inaccurate displacement vector positions, a disparity correction procedure is also available. The calibration module (CalVi) supports accurate calibration with the camera models mostly used in the PIV community: polynomials, rational functions and the pinhole camera model. Moreover, it supports the integration of the pinhole camera model with a refractive correction model for cylindrical geometries and camera calibration procedures working with unknown positions and orientations of the calibration target. In such a way, the target may be moved by hand without losing accuracy in comparison with a conventional calibration, in which the target is displaced with the aid of translational and rotational stages and knowledge of the target positions is strictly required. Already used in numerous experimental studies by the Experimental Thermo-Fluid Dynamics group of University of Naples “Federico II” and international collaborating groups, PaIRS-UniNa offers a rapid and effective free alternative to commercial PIV software for research and industrial use.

New implicit time integration schemes combining high frequency damping with high second order accuracy

It is shown that weighted linear combinations of the generalised-α method and certain related higher order schemes allow for the formulation of unconditionally stable single step time integration methods with improved second order accuracy and more targeted high-frequency damping. It is also shown that, if the user controlled high frequency damping parameter ρ∞ is set to zero, the new schemes can be expressed as linear multistep backward difference formulae and, in a particular case, recover Park's method. The performance of the proposed methods is illustrated in terms of mathematical analysis and a number of linear and nonlinear numerical examples including finite element based solutions of the incompressible Navier-Stokes equations.

Synergistic Nanoarchitectonics: Precision Membrane Engineering for Rare Earth Selective Separation

AbstractRare earth elements (REEs) are vital in high‐tech industries and defense due to their strategic significance. Crafting an efficient membranes channel for REE separation poses a significant challenge. Employing 2‐methylimidazole‐hydrolyzed OH−, dopamine polymerization is initiated and then Zn2+ coordinates with 2‐methylimidazole on PDA surfaces. The confined symbiotic reaction yields 2D vertical heterojunctions of GO/ZIF‐8/PDA (G/Z/P). During separation, partially dehydrated smaller hydrated lanthanide ions preferentially access interlayers, expanding and stabilizing the interlayer space by coordinating with PDA, thus excluding larger hydrated scandium ions from the membranes. Some scandium ions entering interlayer channels are sequestered by N in size‐matched ZIF‐8 pores. This distinctive mechanism facilitates selective scandium separation from other REEs (other REEs/Sc selectivity ≈68.73), achieving nearly complete Sc3+ rejection in a single step. The methodology offers unprecedented insights into precise nano‐space material synthesis, indicating promising strides in advancing scandium production.

The Immediate Effect of the Novel Hybrid Passive Spring Damper Ankle-Foot Orthosis on Spatiotemporal Parameters of Walking in Patients with Multiple Sclerosis

ABSTRACT Introduction Patients with multiple sclerosis (PwMS) experience damage to their central nervous system, which can lead to unilateral or bilateral drop foot. The presently utilized ankle-foot orthoses (AFOs) for PwMS have exhibited limited clinical efficacy. Objective This current study evaluated the immediate effects of the Novel Hybrid Passive Spring Damper Ankle-Foot Orthosis (HPSDAFO) on walking in PwMS. Study design A cohort trial. Methods Twelve PwMS suffering of unilateral drop foot took part in this study. They were given the novel HPSDAFO and underwent kinematic assessment including walking along a 10-m walkway at a self-selected speed, both with and without the HPSDAFO. Results The novel HPSDAFO device significantly changed gait parameters of ankle range of motion, single limb support duration, and step and stride length of the affected side (P < 0001). Furthermore, the device also improved the symmetry index and resulted in increased walking speed and cadence. Conclusions The application of the novel HPSDAFO on the affected side of PwMS resulted in a notable enhancement in the symmetry of spatial parameters, which highlights the need for additional research in this area. Clinical Relevance Using the novel HPSDAFO, PwMS can walk without adverse effects, promoting the normalization of their spatiotemporal walking parameters.

O-190 Hormone-free versus FSH-primed infertility treatment of women with polycystic ovary syndrome using biphasic in vitro maturation: a randomised clinical trial

Abstract Study question Is any hormone stimulation needed to treat infertility in women with polycystic ovary syndrome (PCOS) using in vitro maturation (IVM)? Summary answer Number of matured oocytes and pregnancy outcomes were similar without or with FSH-priming in women with PCOS undergoing IVM. What is known already In vitro maturation (IVM) is a low-intervention alternative to in vitro fertilisation (IVF) for infertility treatment. Current standard clinical practice for human IVM is to prime patients for 2–5 days with follicle-stimulating hormone (FSH) and/or human chorionic gonadotropin (hCG) before harvesting immature or mixed maturity oocytes for infertility treatment. This gonadotropin priming is thought necessary to induce sufficient follicle and oocyte development in vivo to support subsequent oocyte maturation and embryo development in vitro when using a single step (monophasic) oocyte maturation culture system. It is not clear whether hormone-free IVM can generate satisfactory pregnancy and live birth rates. Study design, size, duration This randomised, controlled trial was conducted at an tertiary IVF center, Ho Chi Minh City, Viet Nam. Between January 2023 and June 2023, 120 women (60 per group) were randomised. Participants/materials, setting, methods Eligible women were aged 18–37 years and had PCOS with an indication for biphasic capacitation-IVM (CAPA-IVM). After providing written informed consent, participants were randomised (1:1) to undergo CAPA-IVM with or without FSH-priming. Participants in the FSH-priming group received two days of FSH injections before oocyte pick-up; no FSH was given in the non-FSH group. After CAPA-IVM, day-5 embryos were vitrified for transfer in a subsequent cycle. The primary endpoint was number of matured oocytes. Main results and the role of chance The number [interquartile range] of matured oocytes after CAPA-IVM did not differ significantly between the non-FSH and FSH groups (13 [9; 18] vs. 14 [7; 18]; absolute difference –1 [95% confidence interval –5, 4]). There were also no significant between-group differences in other oocyte and embryology outcomes, including the number of cumulus-oocyte complexes, number of fertilized oocytes, total number of blastocysts and good blastocysts, and total number of frozen embryos. No between-group differences were found in the rates of ongoing pregnancy and live birth, which were both 38.3% in the non-FSH group and both 31.7% in the FSH group; the miscarriage rate at < 12 weeks’ gestation was 5.0% in both groups. Maternal complications were infrequent and occurred at a similar rate in the non-FSH and FSH groups; there were no preterm deliveries before 32 weeks’ gestation. Limitations, reasons for caution The characteristics of the study population (relatively young, lean women with PCOS from Viet Nam) limit the external generalisability of the study findings, and the findings need to be replicated in other populations. Wider implications of the findings Hormone-free assisted reproductive technology (ART) for women with PCOS appears to be feasible when using CAPA-IVM, and does not result in any loss of clinical efficacy. Benefits of hormone-free ART would be a reduction in medical risk, lower cost, fewer monitoring requirements, and greater convenience and flexibility. Trial registration number NCT05600972

In Situ Growth of Lead‐Free Double Perovskite Micron Sheets in Polymethyl Methacrylate for X‐Ray Imaging

AbstractPb‐free double‐perovskite (DP) scintillators are highly promising candidates for X‐ray imaging because of their superior optoelectronic properties, low toxicity, and high stability. However, practical applications require Pb‐free DP crystals to be ground and mixed with polymers to produce scintillator films. Grinding can compromise film uniformity and optical properties, thereby affecting imaging resolution. In this study, an in situ fabrication strategy is proposed to facilitate the crystalline growth of Pb‐free Cs2AgInxBi1‐xCl6 micron sheets in polymethyl methacrylate in a single step. By adjusting the In3+/Bi3+ ratio, Cs2AgIn0.9Bi0.1Cl6/PMMA composite films (CFs) with excellent scintillation properties are obtained, including a light yield of up to 32000 photons per MeV and an X‐ray detection limit of 87 nGyairs−1. This strategy also enabled the production of large Cs2AgIn0.9Bi0.1Cl6/PMMA CFs, which demonstrated favorable flexibility and stability, fabricating products with advanced eligibility for commercial applications. The CFs exhibited outstanding performances in X‐ray imaging, producing high‐resolution structures and providing a new avenue for the development of Pb‐free DP materials in fields such as medical imaging and safety detection.

P-475 Introducing the future of personalized fertility care - AndreiaAI

Abstract Study question Is it possible to build a robust and accurate large language model, from anonymized patient records, to predict live birth occurrence? Summary answer A prototype provides answers in different languages, creates charts/tables based on natural language inputs, and runs statistical analysis, predicting live birth occurrence with 78% accuracy. What is known already The capability for a non-technical person to query clinically valid information and safely managed data represents the single largest step towards the democratization of clinical knowledge to the layperson in the history of ART. Whilst patients are used to Googling their symptoms, Chat GPT was trained on the open internet and will provide equally poor advice. However, when the information is locked down and limited to a specific data set, the ability of a LLM to parse questions as code and return answers specific to the underlying data set presents great opportunities to improve patient and clinician awareness. Study design, size, duration Design: proof of concept Size: 1 million anonymized patient outcome records were gathered from the regulator’s anonymized register (i.e., HFEA, from 1991 to 2018) Duration: between August and December 2023 Participants/materials, setting, methods Participants/materials: 1 million anonymized patient outcome records were gathered from the regulator’s anonymized register (i.e., HFEA, from 1991 to 2018) Method: Data was merged and pre-processed for analysis. As an approach to machine-learning modeling, Ensembling with Random Forest and GB was chosen. Different models were tested to reach maximum accuracy and performance. Patient scenarios based on evidence-based cases were used. A ChatGPT-like interface was preferred as it is simple for doctors and patients to interact with. Main results and the role of chance A prototype model was created, using anonymized patient outcome records, and formulated various patient scenarios based on evidence-based cases, to demonstrate new capabilities. The capabilities include the ability to provide answers in different languages for non-native English speakers, to dynamically create charts and tables based on natural language inputs, and to run sophisticated statistical analysis linked to the underlying dataset for example predict live birth occurrence based on demographic inputs,s and presentation of symptoms. The prototype went as far as creating an ML model to predict live birth occurrence with a 78% accuracy. This was achieved by using an Emsebling method for a binary classification problem (random forest) with live birth occurrence as the target and 14 features, such as age, treatment, and diagnosis. Whilst the technical nature of how this was delivered is extremely important for explainable AI and to engender trust as opposed to replacing the doctor with just another black box, the fact that a predictive model based on 1 million patients and linked back to a specific patient scenario in under 30 seconds for the end-user perspective is proof of capability that personalized insights can be quickly and accurately generated in clinical consultation. Limitations, reasons for caution The study was a proof of concept which needs further research and validation in a clinical setting. Wider implications of the findings The proof of concept evidences the capabilities of LLMs to increase awareness of fertility information. It is accessible, in 40 different languages, on-demand, free to the end-user, and clearly visualized. Access to accurate information that provides personalized insights based on specific criteria, speeds up the time to diagnosis and treatment. Trial registration number n/a

P-234 The internal temperature of the catheter during the transfer: potential effects on the embryonic microenvironment

Abstract Study question Do the external environment and the different loading procedures affect temperature in the transfer catheter and embryo development during embryo transfer? Summary answer The inner temperature of transfer catheter dropped during the embryo transfer. Mouse 2-cell-embryos exposed to the transfer showed lower blastocyst and hatching rates. What is known already Embryo transfer is a crucial process composed of complex phases, which start from embryo loading into the catheter, and culminate in the transfer room in an open environment at room temperature. Although the transfer technique has been refined over time and several adjustments have been proposed towards its standardization (ASRM, 2017, D’Angelo et al., 2022), there is still great variability (Cirillo et al 2020). Few literature data are available regarding possible influences of the external environment on inner catheter conditions. Only recently, temperature drop during transfer has been described (Macklon et al 2021). Study design, size, duration We addressed possible temperature variations to which the embryo is exposed during transfer, by using a thin temperature probe placed inside a catheter. We simulated a total of 120 transfers (60 transfers with “fluid only” methodology and 60 with “air-fluid”). Additionally, we performed Mouse Embryo Assay (MEA) during transfer simulations and then cultured up to blastocyst to assess any impact of temperature drop exposure during transfer Participants/materials, setting, methods 120 mock transfers were performed, recording the temperature in the inner catheter were the embryos are loaded. In our setting, the procedure lasts 60-100 seconds. The internal temperature of the catheter was recorded after 15 min of preheating, at the loading and in the following 60 seconds. Environmental temperature and humidity values were recorded. A total of 73 mouse embryo were used for MEA (39 exposed to catheter, 34 controls) and cultured in time-lapse Main results and the role of chance The 120 simulations took place at an average environmental temperature of 22.43 °C± 0.85, and a relative humidity of 57.4%± 5.1. After 15 min of preheating the empty catheter reached an average temperature of 33.04 °C ±2.08. Upon loading, the average temperature inside the catheter was 31.22 °C± 1.67 and after 60 seconds, it dropped to 29.15 °C ±1.88. The comparison between the “fluid only” and “air-fluid” shows the following results: internal temperature at “fluid only” loading 31.97 °C ± 1.87, “air-fluid” 30.11 °C ± 1.48 (p < 0.001); Average temperature after 60 seconds “fluid only” 29.98 °C ± 1.71, “air-fluid” 27.91 °C ± 1.38 (p < 0.001). Seventy-three 2-cells mouse embryos were thawed and used to perform MEA tests. 39 of them were exposed to 5 transfer simulations of 200 seconds, and then put in culture in time-lapse incubator (single step medium, 6% CO2, 5%O2). The blastocyst rate was 82% in the control group and 74% in the exposed group (X2 (1, N = 73) = 0.6783, p >0.05). Hatching rate significantly differed (61% in controls vs. 34% in the exposed (X2 (1, N = 57) = 3.9, p = 0.04). Morphokinetics were not different among groups Limitations, reasons for caution The monocentric nature of this pilot study can represent the limitation of the single environmental conditions at which the catheters are exposed during the embryo transfer Wider implications of the findings The transfer procedure exposes embryos to a drastic decrease in temperature, directly proportional to the execution time. Mouse embryos showed an impaired development after being exposed to transfer simulations. Novel strategies to stabilize temperature during embryo transfer are needed. Trial registration number non-clinical trial

P-274 Humid or dry culture with medium renewal: impact on A-Grade Day 5 blastocysts. Randomized prospective cohort study

Abstract Study question Could the humid embryo culture impact the development of top-graded blastocysts? Summary answer While no differences were observed in the overall blastocyst development rate, humidity conditions emerged as a significant factor influencing the achievement of A-Grade D + 5 blastocysts. What is known already In recent years, several publications have highlighted the advantages of humid culture (HC) over dry culture (DC) (<45% relative humidity, RH). HC, with its lower risk of media evaporation and consequent osmolality changes. Nevertheless, to the best of our knowledge, this is the first prospective study addressing this aspect through oocytes from the same cohort. Study design, size, duration We have been conducting a randomized control trial (RCT) consisting on 379 MII oocytes from 30 patients from August 2022 until January 2024 (and ongoing). After ICSI procedure, sibling oocytes were divided following 1:1 ratio into both humid (>70% HR) and dry (0% HR) chambers of a Geri incubator (Genea Biomedx, Australia). Ejaculated sperm ICSI cycles with >5 fresh MII were included in the analysis. Time-lapse cleavage timings were manually gathered. Participants/materials, setting, methods Extended culture to the blastocyst stage (D + 5, D + 6 or D + 7) was performed in 80μL of single step G-TL medium (Vitrolife, Sweden). HC remained undisturbed while medium was renewed on D + 3 in DC by changing the culture dish. Fertilization, overall blastocyst development and A-Grade Day 5 blastocysts rates (ASEBIR’s morphology criteria) were compared. Statistical analysis consisted on Chi-squared test, t-student test and logistic regression for computation of Odds Ratio (OR, 95%CI). Main results and the role of chance While no statistically significant differences were observed in the fertilization rate (DC: 76.4%; HC: 69.1%, NS) or the overall usable blastocyst development rate (DC: 48.6%; HC: 53.1%, NS), noteworthy distinctions emerged in the proportion of top-graded embryos based on ASEBIR’s morphology criteria. Specifically, the A-Grade Day 5 blastocyst development rate displayed significant disparities when comparing all fertilized embryos in each culture type (DC: 19.9%; HC: 32.3%; p < 0.05) and also among all usable blastocysts (DC: 40.8%; HC: 60.9%, p < 0,05). The Odds Ratio for achieving A-Grade Day blastocysts depending on type of humidity conditions was 1.87 (1,099 – 3,235) for the humid culture. Moreover, time-lapse imaging facilitated the detection of direct cleavage presence, which was similarly distributed between both incubation types (DC: 27/146; HC: 23/130, NS). However, only 3 embryos showing direct cleavage and cultured in HC reached the blastocyst stage at Day 6, while in dry incubator, 10 embryos with direct cleavage were able to develop to usable blastocysts, three of them being A-Grade on Day 5. Limitations, reasons for caution This study is currently ongoing, aiming to increase the sample size. Future analyses will explore clinical outcomes, especially for underrated embryos, day 6 blastocysts, and those with morphodynamic phenomena. While A-grade blastocyst rate variations may not impact pregnancy outcomes directly, they could influence cumulative success rates. Wider implications of the findings These data highlight that day+3 medium renewal doesn’t equalize conditions to humid culture. Ongoing efforts involve validating these insights through an expanded sample size and extensive outcome analysis. Investigating the long-term impact on clinical outcomes and refining culture protocols can amplify the relevance of these findings in assisted reproductive technologies. Trial registration number pending_protocolOK

P-156 Ultra-fast warming of blastocysts yields better survival rates for D5 non-PGT and similar survival rates for D5/D6 PGT blastocysts

Abstract Study question Can an ultra-fast single-step warming of vitrified blastocysts yield similar survival rates and post-warming development compared to the standard multi-step rehydration protocol? Summary answer Ultra-fast warming, shows significantly better rates of survival and post-warming development in vitro for non-PGT blastocysts and similar rates for PGT blastocysts. What is known already Excellent survival rates have been reported since the introduction of human blastocyst vitrification. Typically, most warming procedures involve three steps with decreasing concentrations of non-penetrating cryoprotectants. Recently, few studies have evaluated the effectiveness of simplifying the procedure to a single step for rehydration, while maintaining comparable survival and pregnancy rates. Adopting simpler procedures can optimize laboratory protocols, workflow, and safety, supporting further advancements in Assisted Reproduction Technologies (ART). Study design, size, duration This validation study ultra-fast warmed 246 vitrified blastocysts [129 non-PGT day5 (cryopreservation period 23/01/2017-24/04/2018 VIT-kit, Fujifilm), 57 PGT day5 (25/01/2023-31/03/2023 VIT-KitNX, Fujifilm), 60 PGTday6 (03/02/2023-22/08/2023 Vit-KitNX, Fujifilm)] and compared survival rates with 379 blastocysts [240 non-PGT day5, 97 PGT day5, 42 PGT day6] vitrified in the same time-periods and warmed for clinical application using the standard multi-step protocol. Ultra-fast warmed blastocysts were cultured for 24h in a time-lapse incubator and subjected to a viability staining. Participants/materials, setting, methods Blastocysts, originating from 111 patients that were donated to validation studies after the legal storage period(n = 129) or deemed non-transferable after PGT(n = 117), were ultra-fast warmed by immersing the straw in a 37 °C thawing solution (1M trehalose; Vit-kitwarmNX; Fujifilm) for 1 minute. Survival rates [total (≥50% intact) and fully (100% intact) ], transfer rates (2h post-warming, ≥50% intact), re-expansion time and the number of damaged cells (Live/Dead kit L-3224, Invitrogen) were analysed. Fisher’s Exact Test (p < 0.05). Main results and the role of chance Overall, the immediate post-warming survival rates (≥50% intact) were comparable between ultra-fast and multi-step warming, 98.4% vs 96.8% respectively (p = 0.3041). However, the fully intact survival rate immediately post-warming was significantly higher with ultra-fast warming (85.4% vs 76.3%; p = 0.0058) as well as the percentage of blastocysts considered transferable (96.7% vs 91.2%; p = 0.0076). Sub-group analysis demonstrated significantly better results for non-PGT blastocysts following ultra-fast warming. Higher immediate survival post-warming (100% vs 96.6%; p = 0.036) and transfer rates (100% vs 90%; p = 0.0002) were achieved. Similar results were observed for PGT blastocysts on both day 5 and day 6, with no significant differences in total, fully intact, and transfer rates observed between the two warming procedures [PGT Day5: 96.5%, 80.7%, and 93.0% vs. 97.9%(p = 0.6270), 69.1%(p = 0.1334), and 93.8%(p = 1.0000)] [PGT Day6: 96.7%, 68.3%, and 93.3% vs. 95.2%(p = 1.0000), 64.3%(p = 0.6761), and 92.8%(p = 1.0000)]. Ultra-fast warmed blastocysts showed a total re-expansion rate of 93.3% in 3.3h±2.7h on average. Viability staining demonstrated very few cells damaged. Zero cell damage was observed in 31.5% of the blastocysts, 59.0% had 1-10 cells damaged, 9.5% >10 cells damaged, and only 4 blastocysts were completely degenerated. Limitations, reasons for caution In order to limit the amount of embryos needed in this validation study, ultra-fast warming outcome parameters were compared with retrospective clinical results of blastocysts vitrified in the same time periods. Ultra-fast warming is a single-step process where precise temperature control(37 °C) of the warming medium is crucial for success. Wider implications of the findings Minimizing handling steps leads to an overall risk reduction in potential exposure to suboptimal warming conditions and operator/handling-related stress. This optimization brings huge efficiency to the busy work schedule that is characteristic for an IVF lab without dropping cryopreservation quality indicators. Trial registration number Not applicable

Novel strategies for the determination of plastic additives derived from agricultural plastics in soil using ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS)

Certain agricultural plastics, i.e., mulching films, are generally considered as potent sources of micro- and nanoplastics (MNPs), due to their direct application on soil and waste mishandling. During the synthesis and fabrication of such agricultural plastics, it is necessary to use chemicals, the so-called plastic additives (PAs), improving the physicochemical properties of the final polymeric product. However, since PAs are loosely bound on the polymer matrix, they can potentially leach into the soil environment with unidentified effects. Clearly, to monitor the fate of PAs in the terrestrial ecosystem, it is necessary to develop accurate, sensitive and robust analytical methods. To this end, a comprehensive analytical strategy was developed for monitoring 16 PAs with diverse physicochemical properties (partition coefficient; −3 < logP<19) in soil samples using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). For this purpose, two different extraction procedures were developed, namely, a single step ultrasound-assisted extraction (UAE) using ethyl acetate or an aqueous solution of methanol and a binary extraction, combining Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) and UAE principles with n-hexane as the extractant. Interestingly, within the sample preparation investigation, we identified in-lab contamination sources of PAs, e.g., centrifuge tubes or microfilters. Such consumables are made of plastic contaminating the procedural blanks and omitting their use was necessary to acquire satisfactory analytical performance. In detail, method validation was performed for 16 compounds achieving recoveries mainly in the range 70–120 %, repeatability (expressed as relative standard deviation, RSD %) < 20 % and limits of quantification (LOQs) ranging between 0.2 and 20 ng/g dry weight (dw). Importantly, the presented strategies are added to the very limited available for PA determination in soil, a topical issue with a significant and rather understudied impact on agriculture.

P-136 Non-invasive PGT (niPGT) using spent culture medium: Relationship between time lapse embryo dynamics and accuracy of niPGT analysis

Abstract Study question Do differences in the morphological characteristics of embryos or the dynamics of the developmental process affect the results of niPGT analysis? Summary answer Embryos with blastomeres that were excluded at compaction showed reduced accuracy of niPGT analysis, but other measured morphological characteristics had no impact. What is known already The PGT-A test, is invasive and requires training to be competent at the technique, aswell as the need for specialized equipment and consumables. In recent years, non-invasive PGT-A (niPGT-A), which analyzes Cell-Free DNA released from cultured embryos, has been attracting attention. Regarding the analysis accuracy of niPGT-A, there are reports that it is equivalent to or even superior to conventional PGT-A. However, at present, it is not clear how methodological differences or embryo dynamics impact results. Study design, size, duration From August 2021 to February 2022, 284 pronuclear-stage frozen embryos (with consent for research use), were thawed and cultured. They were individually cultured for at least 40 hours after Day 4. For the 150 embryos that reached the blastocyst stage, culture medium and the blastocysts were collected and subjected to chromosome aneuploidy analysis by NGS. For the 138 blastocyst with a result, the concordance rate was calculated with that obtained from the culture medium samples. Participants/materials, setting, methods The embryos were individually cultured in a single step medium using a 10 μl droplet from Day 4. The entire culture period was recorded in a time-lapse incubator. The concordance rate for all chromosomes between culture medium sample and the blastocyst was compared to recorded embryo dynamics, e.g. presence or absence of cumulus cell attachment, sperm attachment, fragmentation, blastomeres that excluded at compaction, shrinkage, and hatching. Chi-square test was used to compare the concordance rates. Main results and the role of chance The overall concordance rate for all chromosomes between the culture medium and the blastocysts samples was 46.4%(64/138). In the blastocysts where there were cumulus cells attached or not it was 43.6% (17/39) vs 47.5% (47/99) respectively, cases where there was still a sperm attached or not it was 53.1% (17/32) vs 44.3% (47/106) resepctively and in blastocysts where there had been fragmentation or no fragmenattion it was 46.9%(38/81) vs 45.6％(26/57) respectively. In cases where shrinkage of the blastocyst had been observed vs no shrinkage the respective concordance rates were 44.9% (48/107) vs 51.6% (16/31), whilst in cases where the blastocyst had hatched or not it was 40.6% (13/32) vs 48.1% (51/106) respectively.For the blastocysts where the blastomeres had been excluded at compaction or not it was 35.6％(31/87) vs 64.7% (33/51) respectively.Significant differences were only identified in cases with blastomere exclusion or no exclusion at compaction (P &amp;lt; 0.01). Limitations, reasons for caution In this study, the culture was continued to day 6 regardless of morphology of the embryo on day 5, which differs from the usual clinical culture process. Wider implications of the findings These results are useful in that they may aid in recognising which morphological features of embryos may impact the results obtained from niPGT and hence its reliability for clinical application. The blastomeres that excluded at compaction are clearly important for improving the accuracy of the niPGT analysis in the future. Trial registration number None

Enzymatic Synthesis of Prebiotic Carbohydrates from Lactose: Integration of Osmotic Membrane Distillation with Batch Reactor System

AbstractThis study aimed to enhance galactooligosaccharide (GOS) yield by maintaining high lactose concentration levels using osmotic membrane distillation (OMD) throughout the synthesis. The main problem influencing GOS yield during transgalactosylation is the decrease of lactose concentration over extended reaction periods. By integrating OMD with a continuous stirred batch reactor, water was progressively removed from the reaction medium. Compared to a standard batch reactor, the OMD-integrated system not only increased GOS yield by a maximum of 20.1% but also reduced the time needed for equivalent lactose conversion about 15–90 min. This integration particularly boosted the formation of longer-chain GOS. A consistent GOS yield of 67% was achieved, with up to 28% lactose conversion, surpassing the non-integrated reactor’s performance. The proposed reactor design shows promise for enhancing GOS production efficiency while concurrently concentrating the reaction medium in a single step.

Palladium-Catalyzed Cascade Carbonylation Reaction: Synthesis of Fused Isoindolinones.

A palladium-catalyzed cascade carbonylation reaction of 2-bromo-N-(2-iodophenyl)benzamides with benzylidenecyclopropanes for the synthesis of fused isoindolinone derivatives has been developed. A broad range of 6/5/6/6 tetracyclic isoindolinone products were efficiently prepared in moderate to good yields with diverse substitution. Two carbonyl groups were incorporated into the substrates in a single step with the formation of four carbon-carbon bonds and two carbon-heteroatom bonds.

Efficient and markerless gene integration with SlugCas9-HF in Kluyveromyces marxianus

The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology.

Fluorometric detection of β-carotene with novel green synthesis carbon quantum dot from Asphodelus

Asphodelus-based CQDs were successfully synthesized in a single step by the hydrothermal synthesis method for detection of β-carotene. TEM, XRD, FTIR, XPS, Zeta potential, UV–Vis absorption and fluorescence measurements were taken to structurally and optically characterize the newly synthesized CQDs. It was determined that the characterized CQDs show high selectivity and sensitivity only to β-carotene among 11 different biomolecules. The detection limit of CQDs for β-carotene was calculated as 8.28 µM. It was determined that the new CQDs respond very quickly and stably without interfering with any other biomolecules. The studies in different pH environments exhibited that CQDs can detect β-carotene at all pH studied. Real sample experiments show that β-carotene can be determined accurately and reliably in practical applications with the novel CQDs. It was thought that the results obtained from this study will make significant contributions to the studies on β-carotene detection, which are limited in the literature.

Development of an eco-friendly fluorescent probe for mefenamic acid sensing in pharmaceuticals and biofluids.

Mefenamic acid, renowned for its analgesic properties, stands as a reliable choice for alleviating mild to moderate pain. However, its versatility extends beyond pain relief, with ongoing research unveiling its promising therapeutic potential across diverse domains. A straightforward, environmentally friendly, and sensitive spectrofluorometric technique has been developed for the precise quantification of the analgesic medication, mefenamic acid. This method relies on the immediate reduction of fluorescence emitted by a probe upon interaction with varying concentrations of the drug. The fluorescent probe utilized, N-phenyl-1-naphthylamine (NPNA), was synthesized in a single step, and the fluorescence intensities were measured at 480 nm using synchronous fluorescence spectroscopy with a wavelength difference of 200 nm. Temperature variations and lifetime studies indicated that the quenching process was static. The calibration curve exhibited linearity within the concentration range of 0.50-9.00 μg/mL, with a detection limit of 60.00 ng/mL. Various experimental parameters affecting the quenching process were meticulously examined and optimized. The proposed technique was successfully applied to determine mefenamic acid in pharmaceutical formulations, plasma, and urine, yielding excellent recoveries ranging from 98% to 100.5%. The greenness of the developed method was evaluated using three metrics: the Analytical Eco-scale, AGREE, and the Green Analytical Procedure Index.