Fresh Medium Research Articles

Glioblastoma cells alter brain endothelial cell homeostasis and tight junction protein expression in vitro.

Glioblastoma (GBM) is an aggressive therapy-resistant brain tumour that may impacts the integrity of the blood-brain barrier (BBB). The BBB is a protective barrier of the central nervous system formed mainly by endothelial cells. This study aimed to investigate the in vitro effect of GBM cells on the BBB. Brain endothelial (bEnd.3) cells were used as a model of the BBB. Glioblastoma-conditioned media (CM) was extracted at the 48-h (h) time-point from the U87 GBM cells and diluted to 40% with fresh media. The effect of the U87-CM collected at 48h on bEnd.3 cell growth was evaluated following 48 and 72h of treatment using the xCELLigence system. Additionally, bEnd.3 cell growth was also investigated in a U87 and bEnd.3 co-culture model continuously for 48h using the xCELLigence system. The migration of bEnd.3 cells was assessed following 48 and 72h using the migration scratch assay. The barrier integrity was evaluated continuously for 1h using the transwell permeability, and the tight junction (TJ) protein expression was evaluated using Western blot assay following 48 and 72h. There was a significant decrease in bEnd.3 cell growth following 32h (p < 0.05), 40h (p < 0.01), and 48h (p < 0.001) of treatment with U87-CM, while co-culturing of bEnd.3 and U87 cells increased cell growth following 16h (p < 0.05), 24h (p < 0.001), 32h (p < 0.01), 40h (p < 0.001), and 48h (p < 0.001). The migration of bEnd.3 cells significantly increased following both 24 (p < 0.05) and 48h (p < 0.01) of treatment with U87-CM. The permeability of bEnd.3 cells co-cultured with U87 for 48h was significantly increased (p < 0.05) at the 15- and 30-min time points. Furthermore, the expression of ZO-1 and occludin was significantly increased (p < 0.05) in both bEnd.3 cells treated with U87-CM as well as bEnd.3 cells co-cultured with U87 cells. The current findings suggest that U87 cells alter the integrity of bEnd.3 cells possibly through the secretomes in the CM and through cell-cell interactions in co-culture models. This may assist in the understanding of the mechanisms by which GBM affects the BBB, which may aid in the management thereof.

Development of a Feasible and Efficient In Vitro Rescue Protocol for Immature Prunus spp. Embryos.

The major factors affecting the in vitro immature embryo rescue efficiencies from Prunus persica or P. armeniaca accessions have been identified, along with improving the feasibility. Variations in the woody plant medium (WPM) were used depending on the embryo size. Embryos less than 5 mm long were cultured in WPM supplemented with 1 μM BAP and 1 μM GA3, while embryos bigger than 5 mm long were cultured in hormone-free medium, with or without vermiculite. The environmental in vitro culture conditions consisted of three phases: a (I) stratification at 4 °C during a 3- to 5-month-long period in the dark, followed by (II) growth of germinated embryos at 14 °C for a 4-week-long period, with 12 h light a day, which favors plantlet development, and finally, (III) growth at 24 °C, with 16 h light a day, until the plantlets were acclimatized in the greenhouse. The germination of smaller embryos, at the end of phase I, ranged from 82.2% to 22.1% for apricots and flat peaches, respectively, whereas for bigger embryos, the germination varied from 97.3% to 53.2% for the same species. The embryo germination for peaches and nectarines ranged from 40.1% to 30.3% for smaller embryos, and from 91.9% to 63.0% for bigger embryos. Endo- and epiphytic contamination, affecting from 7.4% to 52.9% of cultured embryos, depending on the fruit type and conservation conditions, and the capacity to acclimate to soil conditions, ranging from 50.4% to 93.2%, were the two most important factors influencing the protocol's efficiency and feasibility. Considering the overall efficiencies, expressed as hardened plants transferred to field plots over clean uncontaminated embryo, the values ranged from 55.8% for nectarines, 54.0% for peaches, 45.6% for apricots, and 23.3% for flat fruits. The addition of vermiculite to the culture medium significantly improved the plantlet development, avoiding subculture to fresh medium when an extension of phase III was required before acclimatization. Compared to laboratory glassware, the use of food glass containers with air-permeable sealing film, along with vermiculite-containing medium, significantly reduced the costs when handling the large number of embryos required for breeding programs.

Membrane-bound receptor for advanced glycation end products (RAGE) is a stable biomarker of low-quality sperm

Abstract STUDY QUESTION Does receptor for advanced glycation end products (RAGE) on the surface membrane of the sperm cell function as a biomarker of low-quality sperm? SUMMARY ANSWER Membrane-bound RAGE at a cellular level directly correlates with low sperm motility, high cell permeability, decreased mitochondrial function, DNA fragmentation, and higher levels of apoptosis. WHAT IS KNOWN ALREADY RAGE has previously been measured by ELISA in low-quality sperm in diabetic men and has been shown to correlate with DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay). STUDY DESIGN, SIZE, DURATION Semen samples were recovered from 60 non-obese, non-diabetic and non-smoking subjects, washed with fresh media, and analysed directly or purified further by differential gradient centrifugation (DGC) or fractionated by direct swim-up before being analysed for sperm motility and molecular health parameters, including cell membrane permeability, cell death, mitochondrial membrane potential, DNA fragmentation, and RAGE protein expression. PARTICIPANTS/MATERIALS, SETTING, METHODS Sperm motility assessments were carried out by computer-assisted sperm analysis (CASA) on 1000 spermatozoa for washed samples and 300 spermatozoa for purified samples. Molecular sperm health parameters were evaluated using flow cytometry with the use of the following markers: DAPI for cell membrane permeability, Annexin V/DAPI for cell death (apoptosis and necrosis), MitoTracker® Red CMXRos for mitochondrial membrane potential, TUNEL assay for DNA fragmentation and 8-hydroxy-2-deoxyguanosine for identification of oxidative damage to sperm DNA, and contrasted to membrane-bound RAGE expression levels, which were evaluated using an anti-RAGE monoclonal mouse antibody. MAIN RESULTS AND THE ROLE OF CHANCE RAGE protein was shown to be present on the acrosomal and equatorial regions of sperm, with the levels of membrane bound receptor strongly correlating with poor sperm health across all parameters tested; motility (R2 = 0.5441, P &amp;lt; 0.0001) and mitochondrial membrane potential (R2 = 0.6181, P &amp;lt; 0.0001) being of particular note. The analysis was performed at a single cell level thereby removing confounding complications from soluble forms of the RAGE protein that can be found in seminal plasma. The expression of the RAGE protein was shown to be stable over time and its levels are therefore not subject to variation in sample handling or preparation time. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Inclusion criteria for this study were non-diabetic, non-obese and non-smoking participants to assess the distribution of RAGE expression in the general population, thereby excluding disease conditions that may increase RAGE expression in sperm or contribute to low sperm quality. The study does not address how RAGE expression may be affected in other patient subpopulations or disease states associated with male infertility. Sperm analysis by flow cytometry is not amenable to the study of males with a low sperm count. WIDER IMPLICATIONS OF THE FINDINGS Results of this study suggest that RAGE expression is a molecular maker of sperm cell health, which may be used for improvements in assisted reproduction through the removal of RAGE expressing sperm and facilitate in the diagnoses of unexplained infertility through its use as a biomarker of male infertility. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by the Irish Research Council under the Government of Ireland Programme (GOIPG/2015/3729) and the Enterprise Ireland Innovation Partnership Programme (IP-2020-0952). All authors declare no competing interests.

Antimicrobial Resistance Development in vitro: Adaptive Laboratory Evolution Method (Review)

INTRODUCTION. High rates of emergence and spread of antimicrobial resistance (AMR) necessitate the rapid development of novel antibacterial medicinal products. The assessment of the microbial potential for AMR development under controlled conditions in vitro can save resources during drug development and marketing authorisation and contribute to creating the most effective medicinal products.AIM. The aim was to determine the possibility of the use of the adaptive laboratory evolution (ALE) method to study the development of antimicrobial resistance.DISCUSSION. A variety of methods can be used to investigate the mechanisms of AMR and the influence of medicinal products on the evolution of bacteria towards AMR. One of the options is the ALE method. ALE experiments are conducted under controlled conditions with prolonged exposure of microorganisms to an antibacterial agent. ALE experiments can include serial transfers of microorganisms to fresh liquid media or Petri dishes, as well as continuous cultivation of microorganisms in a chemostat. ALE protocols are used to develop resistance to different antibacterial agents and require meticulous control of the experimental conditions. To obtain reliable results in an experiment, it is necessary to identify parameters that may affect AMR development in microorganisms. These parameters include but are not limited to the concentration of the antibacterial agent, the number of consecutive passages, the duration of incubation.CONCLUSIONS. To achieve the necessary conditions for resistant microorganisms to form, it is essential to adhere strictly to ALE setup requirements, such as using antibacterial agents at subinhibitory or dynamically increasing concentrations (relative to the minimum inhibitory concentrations for the ancestral strain), performing a certain number of passages for ≥20 generations, and incubating cultures until the stationary phase. Despite the fact that ALE experiments are rather lengthy, these studies can reduce the potential waste of resources on developing new compounds that may have to be discontinued at the stage of production because of AMR development.

Evolution of pH-sensitive transcription termination in Escherichia coli during adaptation to repeated long-term starvation

Fluctuating environments that consist of regular cycles of co-occurring stress are a common challenge faced by cellular populations. For a population to thrive in constantly changing conditions, an ability to coordinate a rapid cellular response is essential. Here, we identify a mutation conferring an arginine-to-histidine (Arg to His) substitution in the transcription terminator Rho. The rho R109H mutation frequently arose in Escherichia coli populations experimentally evolved under repeated long-term starvation conditions, during which the accumulation of metabolic waste followed by transfer into fresh media results in drastic environmental pH fluctuations associated with feast and famine. Metagenomic sequencing revealed that populations containing the rho mutation also possess putative loss-of-function mutations in ydcI, which encodes a recently characterized transcription factor associated with pH homeostasis. Genetic reconstructions of these mutations show that the rho allele confers plasticity via an alkaline-induced reduction of Rho function that, when found in tandem with a ΔydcI allele, leads to intracellular alkalization and genetic assimilation of Rho mutant function. We further identify Arg to His substitutions at analogous sites in rho alleles from species that regularly experience neutral to alkaline pH fluctuations in their environments. Our results suggest that Arg to His substitutions in Rho may serve to rapidly coordinate complex physiological responses through pH sensing and shed light on how cellular populations use environmental cues to coordinate rapid responses to complex, fluctuating environments.

Formulation and evaluation of extended-release floating tablets of labetalol hydrochloride 200 mg

The aim of present work is to formulate and evaluate extended-release floating tablets of Labetalol HCL to improve the bioavailability, Patient compliance and solubility on oral floating drug of Labetalol HCL. Labetalol HCL is mainly used in the treatment of hypertension, which is BCS Class Ⅰ drug i.e. Highly soluble and highly permeable. The tablets were prepared by wet granulation method by using different concentrations of HPMC polymer. The tablets were evaluated for Preformulation characteristics, Pre compression parameters and post compression parameters. In-vitro dissolution studies were performed for all prepared formulations by using dissolution test apparatus employing a paddle stirrer at 50 rpm and 37 ± 0.5°C, 0.1N HCL buffer was used as dissolution medium. Samples of 5ml each were withdrawn at different time intervals. Each sample withdrawn was replaced with an equal amount of fresh dissolution medium. Samples were diluted and assayed at 302 nm using Agilent UV Visible double beam spectrophotometer. The increased drug release is due to the presence of low concentration of HPMC K4 m. The drug release kinetics was calculated for all the formulations, among all the formulations F8 was taken as optimized formulation whose percentage drug release was found to be 96%. It indicates the release follows zero order and Hixon kinetics. Hence formulation F8 was Considered as the optimized batch and stability studies were conducted at 40oc±2oc/75±5%RH, Storage condition for 3 months and no change was observed.

Cell-Free Extracts of the Ginseng Soil Bacterium Pseudomonas plecoglossicida Promote Suppression of Resistance of American Ginseng (Panax quinquefolius) to Root Rot Caused by Ilyonectria mors-panacis.

A prior report showed that soil previously planted with American ginseng (Panax quinquefolius) contained compound(s) which could reduce ginseng resistance to root infection by Ilyonectria mors-panacis, and this was not found in extracts from ginseng roots or soils not previously planted with ginseng. However, the origin of this ginseng-related factor in ginseng soils is unknown. An isolate of Pseudomonas plecoglossicida obtained from soil where P. quinquefolius had been harvested grew more in culture media when ginseng root extract was included, indicating the use of compounds in the extract as nutrients. Treatment with cell-free extracts from media containing ginseng root extracts where P. plecoglossicida had been cultured resulted in root lesions caused by I. mors-panacis being significantly larger than roots treated with fresh media containing root extract or with cell-free media inoculated with the same bacterial isolate without root extract. Levels of ginsenosides in the media decreased over time with incubation. Genome sequencing revealed that the bacterium had genes homologous to those reported for ginsenoside metabolism, which can release sugars for microbial growth. Thus, a ginseng soil bacterium, P. plecoglossicida, can create compound(s) suppressive to root rot resistance, similar to that found in soils previously planted with ginseng, indicating that the activity suppressing root rot resistance in soil previously planted with ginseng may be of microbial origin, utilizing compounds from ginseng roots.

Molecular Diversity Analysis of in vitro and Irradiated Tomato (Lycopersicon esculentum Mill) Grew Under Salt Stress Expressed by SCoT and ISSR Markers

Tomato buds of cv. Idkawy were cultured in vitro on solid MS medium with 0.2 mg-l BAP. The plantlets that were produced were exposed to different doses of gamma radiation, ranging from 100 to 200 Gy. Afterward, single pieces of nodes were cut and moved to a fresh MS medium with 0.2 mg-l of BAP. The gamma radiation caused a mortality rate of 18.75% to 52.5% among the explants. The surviving plantlets were then cut into single node pieces and transferred to an MS medium containing 0.2 mg-l of BAP, with added NaCl concentrations of either 50 or 100 mM. There was increased mortality of the vegetative buds on the explants with increased salt concentrations. It was shown that the all gamma radiation doses caused reduced the percentage of survival at saline levels. The genetic diversity was assessment by using ten primers for each SCoT and ISSR markers to six irradiated treatments grew under salt stress (100 Gy x 50 mM, 150 Gy x 50 mM, 200 Gy x 50 mM, 100 Gy x 100 mM, 150 Gy x 100 mM, 200 Gy x 100 mM). It was showed that the polymorphism percentage mean of SCoT marker (29.56%) is higher than the ISSR marker (26.78%). The average of PIC values for both markers SCoT and ISSR were 0.197 and 0.288 (PIC ˂0.5), as well as, MI values were 0.077 and 0.081, respectively. In contrast, when considering the number of alleles (Ne), Nei's genetic diversity (H), and Shannon's information index (I) parameters, it was observed that the greatest genetic variation was caused by the combined treatment of 200 Gy x 50 mM NaCl using the SCoT marker. On the other hand, with the ISSR marker, the highest induced genetic variation was seen with the combined treatment of 150 Gy x 50 mM NaCl. The obtained results demonstrate that SCoT marker was more accurate and efficient than ISSR marker for distinguishing and genetic variation analysis of irradiated tomato plantlets grew under salt stress. The relationships within treatments were estimated through cluster analysis (UPGMA) based on SCoT and ISSR analysis.

In Situ Live Monitoring of Extracellular Acidosis near Cancer Cells Using Digital Microfluidics with an Integrated Optical pH Sensor Film.

We demonstrate the live monitoring of extracellular acidification on digital microfluidics using a chip-integrated fluorescent pH sensor film. The metabolism of various types of live cells including cancer and healthy cells were investigated through recording the extracellular pH (pHe) change. An optical pH sensor array was integrated onto a digital microfluidic (DMF) interface with a diameter of 2 mm per pH-sensing spot. Miniaturized, label-free, and noninvasive monitoring of extracellular acidosis on DMF was realized within a pH range of 5.0-8.0 with good sensitivity and rapid response. The pH sensitive probe fluorescein-5-isothiocyanate was covalently bound to poly-2-hydroxyethyl methacrylate and immobilized on a circularly exposed indium tin oxide interface on the DMF top plate. The surface of the fabricated pH sensor spots was modified with polydopamine via self-polymerization. Direct cell attachment on the sensor surfaces enabled rapid pH detection near the cell membranes. Automatic medium exchange on cell-attached pH sensing sites was achieved though solution passive dispensing on DMF. The developed DMF platform was used to monitor the pHe decrease during MCF-7 and A549 cancer cell proliferation due to abnormal glycolysis metabolism. A rapid pH decrease at the pH sensing area in the presence of cancer cells could be detected within 2 min after fresh medium exchange, while no obvious pHe change was observed with HUVEC healthy cells. Real-time detection of cell acidification and cellular response to different metabolic conditions such as higher glucose levels or administered anticancer drugs was possible.

Treatment of cryopreserved bovine sperm with calcium ionophore A23187 increases in vitro embryo production

After ejaculation, mammalian sperm undergo a series of molecular events conducive to the acquisition of fertilizing competence. These events are collectively known as capacitation and involve acrosomal responsiveness and a vigorous sperm motility called hyperactivation. When mimicked in the laboratory, capacitating bovine sperm medium contains bicarbonate, calcium, albumin and heparin, among other components. In this study, we aimed at establishing a new capacitation protocol for bovine sperm, using calcium ionophore. Similar to our findings using mouse sperm, bovine sperm treated with Ca2+ ionophore A23187 were quickly immobilized. However, these sperm initiated capacitation after ionophore removal in fresh medium without heparin, and independent of the Protein Kinase A. When A23187-treated sperm were used on in vitro fertilization (IVF) procedures without heparin, eggs showed cleavage rates similar to standardized IVF protocols using heparin containg synthetic oviduct fluid (IVF-SOF). However, when A23187 pre-treated sperm were further used for inseminating eggs in complete IVF-SOF-heparin, a significantly higher percentage of embryo development was observed, suggesting a synergism between two different signaling pathways during bovine sperm capacitation. These results have the potential to improve current protocols for bovine IVF that could also be applied in other species of commercial interest.

Outdoor semi-continuous cultivation of Synechococcus sp. for enhanced carotenoid production

The carotenoid productivity of a euryhaline Synechococcus sp. was studied in indoor and outdoor conditions. Indoor experiments revealed that the strain could maintain growth and carotenoid accumulation within a salinity range of 2.5–8.0 % NaCl. In an indoor two phase semi-continuous cultivation in PBRs, every three days, 50 % of the culture was replaced with fresh growth media in phase one, whereas the removed culture was grown for another three days in the second phase to enhance the carotenoid yield. Biomass and carotenoid productivity of the strain in the second phase were similar in five consecutive cultivation cycles. Next, the same two-phase cultivation experiment was conducted in 5 sq. m outdoor raceway tanks. However, for the outdoor experiment, carotenoid-rich biomass was harvested from the second phase by membrane filtration, and the growth media was recycled for cultivating in the first phase. The salinity of the culture continued to increase as the evaporated water was balanced by seawater. The semi-continuous cultivation of the first phase continued for 20 days before the culture salinity reached 7.4 % NaCl. For Synechococcus sp., the growth and carotenoid productivities were not affected within this salinity range.

Chlorella vulgaris cold preservation (4∘C) as a means to stabilize biomass for bioreactor inoculation: A six-month study

Chlorella vulgaris cells were maintained over six months (or tentatively) using three protocols: two-week subculturing (positive control), storage at 4∘C, and simple abandonment (negative control). Cultures were monitored by their optical and cell densities over the trial period. Cells were characterized by their size, pigment profile, photosystem II status (OJIP test), electron transport rate assay (light curve), and lag phase duration when regrown. The abandoned cultures quickly showed cells deviating from their nominal state (increased size, a loss of their pigments, a negative alteration of their photosynthetic capacity, and an extended lag phase when inoculated into fresh medium). Frequent subculturing yielded reasonably stable performances. Yet, our experience showed that uncontrollable factors (human errors, lack of communication between teams) could expose the cultures to unfortunate incidents. 4∘C preservation allowed the cells to have a constant size and a slightly increased, yet stable, pigment profile associated to a dark acclimation (+12 % total chlorophyll). Finally, regrowth tests demonstrated that 4∘C preservation induces slightly improved performance (lag phase duration reduced by 9.5 %) than frequent subculturing. Those findings advocate for the use of 4∘C preservation to reduce cell maintenance work and conserve a pool of cells in a similar state to be used as repeatable inoculum for larger-scale experiments while nullifying otherwise batch-to-batch variation effects. Subculturing work can be reduced from once every two weeks to once every six months at least.

Establishment of a semi-continuous scale-down clone screening model for intensified perfusion culture.

Perfusion cultures have been extensively used in the biotechnology industry to achieve high yields of recombinant products, especially those with stability issue. The WuXiUP™ platform represents a novel intensified perfusion that can achieve ultra-high productivity. This study describes a representative scale-down 24-deep well plate (24-DWP) cell culture model for intensified perfusion clone screening. Clonal cell lines were expanded and evaluated in 24-DWP semi-continuous culture. Cell were sampled and counted daily with the aid of an automated liquid handler and high-throughput cell counter. To mimic perfusion culture, 24-DWP plates were spun down and resuspended with fresh medium daily. Top clones were ranked based on growth profiles and productivities. The best performing clones were evaluated on bioreactors. The selected clones achieved volumetric productivity (Pv) up to 5g/L/day when expressing a monoclonal antibody, with the accumulative harvest Pv exceeding 60g/L in a 21-day cell culture. Product quality attributes of clones cultured in 24-DWP were comparable with those from bioreactors. A high seeding strategy further shortened the clone screening timeline. In this study, a 24-DWP semi-continuous scale-down model was successfully developed to screen for cell lines suitable for intensified perfusion culture.

The impact of implementing a non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) embryo culture protocol on embryo viability and clinical outcomes.

Are modifications in the embryo culture protocol needed to perform non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) affecting clinical reproductive outcomes, including blastocyst development and pregnancy outcomes? The implementation of an embryo culture protocol to accommodate niPGT-A has no impact on blastocyst viability or pregnancy outcomes. The recent identification of embryo cell-free (cf) DNA in spent blastocyst media has created the possibility of simplifying PGT-A. Concerns, however, have arisen at two levels. First, the representativeness of that cfDNA to the real ploidy status of the embryo. Second, the logistical changes that need to be implemented by the IVF laboratory when performing niPGT-A and their effect on reproductive outcomes. Concordance rates of niPGT-A to invasive PGT-A have gradually improved; however, the impact of culture protocol changes is not as well understood. As part of a trial examining concordance rates of niPGT-A versus invasive PGT-A, the IVF clinics implemented a specific niPGT-A embryo culture protocol. Briefly, this involved initial culture of fertilized oocytes following each laboratory standard routine up to Day 4. On Day 4, embryos were washed and cultured individually in 10 μl of fresh media. On Day 6 or 7, blastocysts were then biopsied, vitrified, and media collected for the niPGT-A analysis. Six IVF clinics from the previously mentioned trial were enrolled in this analysis. In the concordance trial, Clinic A cultured all embryos (97 cycles and 355 embryos) up to Day 6 or 7, whereas in the remaining clinics (B-F) (379 cycles), nearly a quarter of all the blastocysts (231/985: 23.5%) were biopsied on Day 5, with the remaining blastocysts following the niPGT-A protocol (754/985: 76.5%). During the same period (April 2018-December 2020), the IVF clinics also performed standard invasive PGT-A, which involved culture of embryos up to Days 5, 6, or 7 when blastocysts were biopsied and vitrified. In total, 428 (476 cycles) patients were in the niPGT-A study group. Embryos from 1392 patients underwent the standard PGT-A culture protocol and formed the control group. Clinical information was obtained and analyzed from all the patients. Statistical comparisons were performed between the study and the control groups according to the day of biopsy. The mean age, number of oocytes, fertilization rates, and number of blastocysts biopsied were not significantly different for the study and the control group. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate, or ongoing pregnancy rate (≥12 weeks) in the study group compared to the control group when stratified by day of biopsy. The limitations are intrinsic to the retrospective nature of the study, and to the fact that the study was conducted in invasive PGT-A patients and not specifically using niPGT-A cases. This study shows that modifying current IVF laboratory protocols to adopt niPGT-A has no impact on the number of blastocysts available for transfer and overall clinical outcomes of transferred embryos. Whether removal of the invasive biopsy step leads to further improvements in pregnancy rates awaits further studies. This study was funded by Igenomix. C.R., L.N.-S., and D.V. are employees of Igenomix. D.S. was on the Scientific Advisory Board of Igenomix during the study. ClinicalTrials.gov (NCT03520933).

An investigation of the dose-dependent safety of bemiparin sodium: A cell culture study

Aim: Fibroblasts are common cells in subcutaneous tissue and they have an important role on healing process. Although patients who are receiving first-generation low molecular weight heparins are more likely to experience skin responses, there is no study evaluating the effect of bemiparin sodium on subcutaneous fibroblasts following administration. In the present study, we aimed to evaluate the effects of bemiparin sodium on fibroblast cell viability in cell culture model. Material and Methods: The L929 cells were incubated for 12 hours in 96-well culture dishes. Fresh medium containing different concentrations of bemiparin was added in five different concentrations (Dilution I: 3,500 IU; Dilution II: 1,750 IU; Dilution III: 875 IU; Dilution IV: 437.5 IU; Dilution V: 218.75 IU). A control group was established with drug-free culture medium. Cell viability analysis was performed after 48 hours of incubation by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The change in cell morphology was examined at each dilution by direct and acridine orange/propidium iodide staining. Results: The highest cytotoxic effect was observed in dilution I compared to the control group (p&lt;0.05). There is a clear deviation from the fibroblastic morphology of the cells in dilution I. The cells have a round morphology with degeneration and nuclear condensation. Conclusion: Although it seems that high doses of bemiparin may have a negative effect on fibroblast cells in subcutaneous tissue, it can be considered that the cytotoxic effects of high concentrations of the drug at the application site will not be clinically significant due to using a single dose per day.

Gray mold disease on bottle gourd caused by Cladosporium tenuissimum in China.

Bottle gourd [Lagenaria siceraria (Mol.) Stand] is a widely cultivated succulent crop species. In December 2022, a serious bottle gourd disease occurred in the protected vegetable planting base of Xingguo County, Ganzhou City, Jiangxi Province, China, with 85% of the 2,100 plants having gray mold disease-like symptoms, including gray spots on the infected fruit. They quickly expanded at suitable temperature and humidity, forming a gray mold layer with inward depressions, which spread to the fruit stem causing watery rot, and the flesh turned black and started to rot. To isolate the pathogen, fruits of the diseased plants were surface-disinfected with 75% ethanol for 30 s, immersed in 0.1% HgCl2 for 1min, rinsed thrice with sterile water, and cultured on a potato-dextrose agar (PDA) medium at 28°C. Mycelia from the diseased tissue were subcultured on fresh PDA medium to obtain pure cultures. After incubation at 25°C for 7 days, olive-green colonies (~2.5mm·d-1) developed. Cultures developed numerous elliptical and limoniform conidia measuring 2.69~9.79 μm to 2.10~5.92 μm (average 5.62×3.12 μm) (n=20). The morphological characteristics of the pathogen resembled those of Cladosporium spp. Fungal genomic DNA was extracted, and the internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (TEF-1α), and actin (ACT) regions were amplified with primers ITS1/4, TEF-728F/986R, and ACT-512F/783R, respectively, and sequenced (Bensch et al. 2012; Jo et al. 2018). Basic Local Alignment Search Tool analysis (BLAST) revealed that the ITS (accession no. OQ186729), ACT (OQ240962), and TEF-1α (OQ240963) sequences of isolate hjt4 shared the highest similarity (99-100%) with those of Cladosporium tenuissimum (accessions no. OM232068, OM256530, OM256526) (Duccio et al. 2015). A phylogenetic tree of the isolate hjt4 and its close relatives within Cladosporium was constructed using the MEGA X neighbor-joining method. The pathogen was identified as C. tenuissimum based on morphological and molecular characteristics. A specimen (JXAU-H2022982) was deposited at the Herbarium of the College of Agronomy, Jiangxi Agricultural University. To confirm its pathogenicity, seven-day-old healthy bottle gourd fruits were disinfected with 75% ethanol, 1 mm-deep wounds were made with sterilized scalpels, and the plants were inoculated with PDA plugs (0.8 cm in diameter) containing actively growing mycelia of isolate hjt4. Plants inoculated with sterile PDA plugs served as controls. Each group contained three fruits, and the experiment was performed in triplicate. All fruits were incubated in a biochemical incubator at 28°C. After 3 days, the fruit surface shrank, and the flesh turned to a black colour and rotten, which rapidly spread to the branches. Control fruits did not develop any symptoms. Reisolated colonies showed the same morphological traits as those of the inoculation isolates, whereas no target colonies were isolated from the control fruits. The pathogen was previously reported to cause leaf blight disease in Coriandrum sativum (Zhou et al. 2022) and sooty spots on Cape gooseberry (Miyake et al. 2022), among others. To our knowledge, this is the first report of gray mold disease caused by C. tenuissimum on bottle gourd in China. The findings provide an important foundation for monitoring and controlling the spread of this disease.

A cell targeting and sorting approach based on the magnetophoretic capturing for early prognostics of metastatic cervical cancer cells

HeLa cervical cancer cells are immortal with telomerase activity and metastatic characteristics similar to circulating tumor cells (CTCs). Here, we report aptamer-modified multilayered magnetic beads (Apt@MBs) that efficiently targeted and captured HeLa cells up to a low concentration of freshly prepared cell suspension (500 cells/mL). Apt@MBs were functionalized with fluorophore-conjugated AS1411-aptamer on an outer layer made up of molybdenum disulfide (MoS2) to target nucleolin on the cell surface of captured HeLa cells. Moreover, this outer MoS2 layer of MBs was nanoporous and could load anticancer drugs inside its porous cavities with the possibility of killing the captured and metastatic CTCs in vivo. An internal core layer of Apt@MBs consisting of Ag–Fe3O4 magnetic particles (MPs) was designed for magnetic manifestations and cell sorting with the possibility of screening CTCs (in the patient's blood samples) for early diagnosis of metastatic cancers. The Apt@MBs after cell capture gave rise to the heavier HeLa-MBs composites to get settled down under gravitational/inertial forces to the bottom of the tube quicker than the free cells (within 10 min). The gravitational settling of HeLa-MBs was further coupled with exposing a magnetic field to effectively capture and enrich the cells at the bottom of the tube (from 91 to 98 % cells). While the fluid containing dead, non-cancerous, or uncaptured cells in the supernatant layers were easily removed by pipetting. The HeLa-MBs after sorting out were resuspended into a fresh culture medium for further incubation or cellular analysis. Moreover, both cisplatin (CP) and epirubicin (EP) loaded Apt@MBs showed the killing of about 50 % of the captured cells. Therefore, we are confident that Apt@MBs can contribute to enumerating patients' blood samples for screening CTCs to timely and efficiently detect metastatic cancers along with the ability to effectively perform prognosis, and treatment of metastatic cancers.

P-138 Effect of biofeedback from the blastocysts on the cumulative pregnancy rate

Abstract Study question The study is to compare cumulative pregnancy rates of embryos transferred with and without spent culture media to confront biofeedback’s role in clinical outcomes Summary answer Transferring the blastocysts with spent culture media improved the pregnancy rates. Biofeedback was observed from the blastocyst stage. The blastocyst freezing and transfer approach improved. What is known already Biofeedback is often encountered in regulating menstrual cycles and other events in continuing gestations with estrogen, progesterone, beta hCG..etc. A similar biofeedback is possible with growing blastocyst for implantation. Since the in-vitro culture of embryos is devoid of its occurrence, it may lead to asynchrony of subsequent events and implantation. There is no scientific evidence from metabolomics on the characterization of these substances or secretions from spent culture medium that includes cell-free DNA giving feedback and its role in improving clinical outcomes. Study design, size, duration This retrospective multi-centric study included the 3591 frozen embryo transfers from 1st Apr 2022 to 31st Nov 2023 out of 4623 women who underwent oocyte retrieval from 1st Jan 2022. Patients were divided into study (group A) and control (group B). In group A, 1035 women received the transfers from spent culture media; in group B, 2556 women received transfers after shifting the blastocysts to fresh culture media before loading into the ET catheter. Participants/materials, setting, methods All the transfers considered in this study were day-5 blastocysts, irrespective of their frozen state. Day-5 frozen-thawed were incubated short-time (∼1 hour) before the transfer. Whereas, the frozen embryos and morulas were cultured till day 5. In the study group, all cultures were conducted in 100ul of culture media under oil per embryo to maintain consistency amongst the group. The rest of the laboratory and clinical procedures were consistent between the two groups. Main results and the role of chance The cumulative pregnancy rate was significantly higher in group A than in group B (60.9% Vs 55.6%; OR = 1.23; 95% CI, 1.07 -1.44, Z-score 2.901, P &amp;lt; 0.003). The effect of spent culture media was further observed by regrouping the day-3 or 4 frozen (group A1) and day-5 frozen patients (group A2). Day-5 thawed blastocysts transferred after short incubation to allow the secretions to directly deposit on the endometrium. Group A1 might have a high concentration of secretions due to its accumulation from the embryo or morula stages. Pregnancy rates in group A1 were higher than A2 but not statistically significant (61.7% Vs 59.7%; OR = 1.08, 95% CI, 0.84-1.4, Z-score 0.654, P = 0.51). The less impact indicates that the feedback came from blastocysts rather than embryos. Limitations, reasons for caution It is not evident how these secretions contribute to biofeedback. Quantification, identifying their time of release and recovery from the qualitative tests for their therapeutic use are the limitations. Nevertheless, spent media can be employed if found beneficial. Wider implications of the findings We have taken a large possible data set for control against the test group and took cumulative pregnancy rate as the primary outcome indicator to prove biofeedback from blastocysts can improve the clinical outcomes irrespective of the age and other factors of infertility. Trial registration number Not applicable

P-533 Impact on clinical outcomes of day-6 extended culture and reduced culture media volume applied to non-invasive PGT-A

Abstract Study question Impact on clinical outcomes of day-6 extended culture and reduced culture media volume applied to non-invasive PGT-A Summary answer The implementation of changes in culture conditions to accommodate niPGT-A has no impact on blastocysts viability or pregnancy outcomes. What is known already The recent identification of embryo cell-free DNA (cfDNA) in the culture media has created the possibility of a non-invasive approach for aneuploidy testing in blastocysts. Concerns have however arisen at two levels. Firstly, the concordance rates that cfDNA results have with the full blastocyst or a trophectoderm (TE) biopsy and secondly, the impact on clinical outcomes of the changes in the embryo culture protocol that are required to implement niPGT-A in the IVF laboratory. Concordance rates of niPGT-A to invasive PGT-A have gradually improved, however the impact of culture protocol changes is not as well understood. Study design, size, duration Six IVF clinics participating in a trial examining concordance rates for invasive and non-invasive PGT-A on day-6/7 blastocysts were involved (Rubio et al., 2020). Patients were recruited from April 2018 to December 2020. In the study group, 428 PGT-A cases following the niPGT-A protocol for embryo culture were included. The control group consisted of 1,392 regular PGT-A cases that did not participate in the study and underwent the standard PGT-A culture protocol. Participants/materials, setting, methods In the control group, embryos were biopsied and vitrified on day 5, 6 or 7. In the study group, niPGT-A culture conditions started on day 4, when embryos were washed and cultured individually in 10μl of fresh media. On day 6/7 blastocysts were biopsied, vitrified and media collected for niPGT-A analysis. Clinic A cultured all embryos up to day 6/7, whereas in clinics B-F a minority of day-5 blastocysts were biopsied and transferred. Main results and the role of chance Statistical comparisons were performed between the study and the control groups. Mean female age, number of oocytes, fertilization rates and number of blastocysts biopsied were not significantly different for the study and the control group. Overall, the niPGT-A protocol did not have any detrimental effect on blastocyst quality, euploidy rates or informativity rates. Deferred single embryo transfers (SET) were performed in both groups. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate or ongoing pregnancy rate (OPR, ≥12 weeks) in the study group compared to the control group when stratified by day of biopsy. Clinic A showed no difference in OPR per transfer regardless of culture protocol (62.3% in control group day 5/6/7; 60.8% in control group day 6/7; and 65.6% in study group day 6/7). For the remaining clinics (B-F), a comparison was made separately between day-5 and day-6 blastocysts that were transferred and cultured according to the standard and to the niPGT-A protocol. No significant differences in OPR were observed, neither between control group day 5 (60.6%) and study group day 5 (58.7%), nor between control group day 6 (44.7%) and study group day 6 (41.9%). Limitations, reasons for caution The limitations are intrinsic to the retrospective nature of the study, and also to the fact that the study was conducted in invasive PGT-A patients and not in niPGT-A cases alone. Wider implications of the findings This study shows that changing current IVF laboratory practice to adopt niPGT-A protocols has no impact on the number of blastocysts available for transfer and overall clinical outcomes. Whether removal of the actual invasive biopsy step leads to further improvements in pregnancy rates awaits further studies. Trial registration number ClinicalTrials.gov (NCT03520933)

Clay Properties in the Mukdadiya Formation for Drilling Fluid Production from Shorau and Shewsoor, N Kirkuk, Iraq

The present study involves assessing the potential of the clay layers in the Muqdadiya Formation (late Miocene–Pliocene) for use in oil well drilling fluids. It is achieved through conducting various tests on the samples under investigation and comparing the results with the standard specifications for drilling fluids set by the American Petroleum Institute (API). The study also compares the effectiveness of different additives in improving the local clays and the impact of adding XC-polymer on the current wicking values. It has been found that XC-Polymer possesses superior properties compared to other polymers when used in the same concentrations. This substance is extremely important due to its direct correlation with the operation of drilling fluid. The test was used to determine that the two research models (Shorau and Shewasoor) at their usual concentrations did not produce the necessary characteristics of the drilling fluid in accordance with API requirements. Appropriate specifications for the drilling fluid were obtained by conducting a series of laboratory tests, increasing the model's concentration to 25 grams, and adding high-viscosity CMC and XC-Polymer to the sample in order to assess the current properties of the two study models in fresh media. The Shurao (M1) and Shewsoor (M2) models did not show current properties suitable for drilling fluid in salty media. After adding XC-Polymer to 25 grams of the models activated with 4 grams of Na2CO3, the two study models showed a response to the activation process and obtained results for current properties suitable for drilling operations in salty media. It turned out that the Shewsoor model had more appropriate specifications than the Shorau model.