Total Cell Number Research Articles

Reduction Disintegration Behavior and Crystallographic Transformation of Vanadium-Titanium Magnetite Pellets during Hydrogen-based Reduction

：There are many advantages of the smelting of vanadium-titanium magnetite pellets by hydrogen-based shaft furnace pre-reduction and electric arc furnace process, including high reduction efficiency, low carbon dioxide emission and high recovery of titanium and so on. The preparation of qualified vanadium-titanium magnetite pellets with low reduction disintegration index (RDI) is prerequisite of this process. The reduction disintegration behavior of vanadium-titanium magnetite pellets during hydrogen-based reduction was systematically investigated in this work, and the crystallographic transformations during the reduction were dig into as well. The RDI+6.3 mm of vanadium-titanium magnetite pellets reached 43.28% after 60 minutes of reduction at 500 °C in a Midrex atmosphere (36% CO, 5% CO2, 55% H2, and 4% CH4). During the reduction of trigonal titano-hematite [(Fe,Ti)2O3] to cubic titano-magnetite, both the single unit cell volume and the total number of unit cells increased dramatically. The total titanium-iron oxide cell volume expanded nearly sevenfold, from 8.75×1022 ų to 5.99×1023 ų, causing considerable pellet swelling. Conversely, the reduction of rhombohedral Fe2TiO5 to trigonal FeTiO3 resulted in a decrease in cell volume. Studying the crystalline transformation is crucial for developing effective methods to inhibit low-temperature reduction disintegration, thereby enhancing the comprehensive utilization of vanadium-titanium magnetite resources.

Enhanced Cell Growth and Astaxanthin Production in Haematococcus lacustris by Mechanostimulation of Seed Cysts

The slow growth and complex life cycle of Haematococcus lacustris pose significant challenges for cost-effective astaxanthin production. This study explores the use of microfluidic collision treatment to stimulate the germination of dormant seed cysts, thereby improving photosynthetic cell growth and astaxanthin productivity in H. lacustris cultivated in well plate and flask cultures. The flow rate (1.0–3.0 mL/min) and the number of T-junction loops (3–30) were optimized in the microfluidic device. Under optimal conditions (a flow rate of 2.0 mL/min with 10 loops), the total cell number density in well plate cultures increased by 44.5% compared to untreated controls, reaching 28.9 ± 2.0 × 104 cells/mL after 72 h. In flask cultures, treated cysts showed a 21% increase in astaxanthin productivity after 30 d, reaching 0.95 mg/L/d, due to higher biomass concentrations, while the astaxanthin content per cell remained constant. However, excessive physical collision stress at higher flow rates and loop numbers resulted in reduced cell viability and cell damage. These findings suggest that carefully controlled cyst mechanostimulation can be an effective and environmentally friendly strategy for Haematococcus biorefining, enabling the production of multiple bioactive products.

SURG-48. LASER INTERSTITIAL THERMAL THERAPY REMODELS THE TUMOR IMMUNE LANDSCAPE IN A MOUSE GLIOMA MODEL

Abstract BACKGROUND Laser interstitial thermal therapy (LITT), a minimally invasive surgical technique for tumor ablation, is increasingly being used to treat primary and recurrent glioma. While the primary goal of using LITT has been to destroy tumor tissue, little is known about the impact of LITT on the overall immune response. To investigate changes in the tumor immune landscape, we utilized a mouse model of LITT and applied it to a poorly immunogenic mouse model of glioma. METHODS SB28 glioma cells were implanted in the right frontal lobe of C57BL/6 mice, and on day 7, an optical fiber is inserted into the tumor, paired with a thermocouple for real-time temperature monitoring. A 30W laser was activated in pulses to maintain a peri-lesional temperature of 44C for 150-180 seconds. The tumor and LITT-associated immune response was analyzed by multiparameter flow cytometry, single cell RNA-seq, and multiplex immunofluorescence. RESULTS LITT (versus sham control) triggered an increase in neutrophils in the tumor during the acute phase at day 3 following treatment. Total immune cell numbers increased over time in both sham- and LITT-treated mice, but there was a significant increase at post-LITT day 7 in select adaptive immune cell populations including type 1 conventional dendritic cells, NK cells, and gamma delta T cells. Monocytes/macrophages comprised the majority of the immune infiltrate, and multiplex immunofluorescence revealed these populations localized to the perimeter of the LITT lesion. scRNA-seq also revealed enrichment of a Fabp5- and Gpnmb-expressing macrophage subset following LITT. CONCLUSIONS LITT modifies the tumor immune infiltrate with increases in neutrophils as well as select adaptive cell and macrophage subsets. Future studies will explore adjunct therapies to further augment the immune response in the setting of LITT.

Comparison of different histomorphological grading systems in vulvar squamous cell carcinoma.

Histopathological biomarkers of carcinomas and their prognostic relevance, such as Broder's grading system (based on the total number of undifferentiated cells) or Bryne's grading system (rating morphological features at the tumor invasive front), have been repeatedly and successfully put to test. Since most studies focus on head and neck cancers or oral carcinomas, for squamous cell carcinoma of the vulva, no standardized and agreed on pathological tumor grading system, yielding prognostic significance, could be determined so far. To determine prognostic associations of different grading systems with regard to groin lymph node metastasis, 73 cases of vulvar carcinomas (VC) were re-examined within our study and Broder's and Bryne's grading system individually performed. To sub-classify between HPV-associated or HPV-independent VC, immunohistochemical p16 stainings were performed. Statistical relationships were evaluated using Spearman correlation and logistic regression analysis, validation was achieved by employment of the likelihood ratio test (LRT) and assessment of ROC curves/AUC values. Within our cohort, Broder's grade I (40≈55%) and Bryne's grade II (48≈66%) were the most frequently assigned histological gradings. We determined a positive correlation of Bryne's grading with the extent of lymph node involvement in HPV-associated tumors and demonstrated the feasibility of Bryne's grading to predict the presence of carcinoma cells within groin lymph nodes (LRT p = 0.0066; AUC value≈0.91) in this cohort. On the other hand, our data suggest that especially HPV-independent tumors may not sufficiently be characterized by current standardly performed grading approaches. Since only Bryne's grading system correlated positively with lymph node involvement in HPV-associated squamous cell carcinoma of the vulva, we propose to include it by name next to the distinct tumor entity on the histopathological report, allowing not only the interpretation of its prognostic relevance but also future research attempts.

Influence of saponin tauroside Sx1 on lifespan and expression of vitamin D receptors in the liver tissue of mice with influenza infection

Introduction. Respiratory infections, including influenza, are often accompanied by hepatitis in humans which pathogenesis is not fully understood. According to the available datan D deficiency is presumably a risk factor in the occurrence of acute respiratory viral infections due to its modulation of immune response. Recent studies indicate that several plant compounds can interact with vitamin D receptors (VDRs) and modulate the activity of VDRs. The biologically active components saponins have found widespread use in clinical practice due to their wide range of biological and pharmacological effects which mechanisms are still largely unclear.Aim. To study the effect of oral administration of saponin tauroside Sx1, obtained from Crimean ivy, on life expectancy and activation of vitamin D receptors in the liver of mice during experimental viral infection of varying severity.Materials and methods. The 11 subgroups were formed from male BALB/c mice, were used in the experiment depending on the infectious dose of influenza virus, 5 LD50 virus or 10 LD50 virus, respectively, including control. Saponin tauroside Sx1 was used as a corrector. Immunohistochemical studies were carried out automatically in a BOND-MAX immunohistainer (Leica, Germany). Primary rabbit polyclonal antibodies to the vitamin D receptor were used.Results and discussion. Due to the administration of tauroside Sx1 at a dose of 200 μg/mouse/day increases the average life expectancy of animals receiving saponin by 4.6 days. Reducing the infectious dose of IV from 10 LD50 to 5 LD50 also changed the onset of death of animals by 2 days in both groups. With different infectious doses of the virus, on the 10th day of the experiment in the subgroups, the expression of vitamin D receptors changes without correction. In subgroup 2V, the number of total positive cells was lower than the control group. Moreover, in the 2Vir subgroup, VDR expression was significantly higher than the control group.Conclusion. The saponin tauroside Sx1 at a dose of 200 µg/mouse/day has a fairly pronounced antiviral effect during experimental infection of mice with the influenza A/H1N1 virus, which is manifested by an increase in the average life expectancy of animals (for 4.6 days) and a decrease in the mortality rate during severe influenza infection, compared with the control group, where 100 % mortality was observed by the 14th day of the experiment. The introduction of saponin on the 4th day of the experiment in all subgroups reduces the total number of immune cells that intensively express VDR.

Effects of calcium lactate on in vitro fertilization and embryonic development in cattle.

Growing demand for embryo transfer is steadily expanding and further studies on in vitro fertilization of cattle. To assess the effect of calcium lactate by replacing Tyrode's Albumin Lactate Pyruvate (TALP) medium composition during fertilization and embryonic development. Sodium lactate and CaCl2 were replaced with 2.0, 3.0, 4.5 mM calcium lactate for TALP medium during fertilization in Experiment 1. In Experiment 2, the concentrations of sodium lactate and CaCl2 were re-modified as control, in comparison with the same concentration of calcium lactate at 4.5 mM. Zygotes were moved to sequential media to match early-and late-stage environments. Embryonic development was examined on day 8 after insemination. 4.5 mM calcium lactate enhanced the rate of fertilization and blastocyst formation (p<0.0001, p<0.01, respectively). It represented differences in the ROS (p<0.01) and GSH levels (p<0.05) and increased blastocyst diameter and total cell number (p<0.05). In Experiment 2, fertilization (p<0.05) and blastocyst formation rates (p<0.01) were increased in 4.5mM calcium lactate under same concentration effect of sodium lactate and CaCl2. Additionally, it reduced the ROS (p<0.01) and increased the GSH levels (p<0.05), leading increase embryo quality. The replacement of calcium lactate in TALP medium enhances fertilization and embryonic development while also improving oxidative stress. Specifically, it has been determined that a concentration of 4.5 mM calcium lactate is the most effective, irrespective of the varying concentrations of sodium lactate and CaCl2. This study presents a novel formulation of a modified TALP medium intended for implantation withing the bovine embryo industry. The current implications of the study are discussed in relation to previously stated objectives and hypotheses.

Identification of Some Fungi Causing Skin Infection and its Relationship with Some Blood Test of Tinea Patients in Tikrit City

Dermatophytes infection is a common problem worldwide and frequent in Iraq. Several reports and articles were published on prevalence, distribution, causes and treatment of dermatophytosis. The current study included the collection of 100 clinical samples (70 skin peels, 20 hair samples and 10 nail samples), as well as 50 blood samples from patients with dermatophytes and skin consultants in Tikrit City and 20 healthy blood samples for the period from November 2023 to March 2024. The results showed that the most common dermatophytes isolated was Microsporum canis and T.rubrum 10 (20 %) isolates from 50 total , followed by M.gypseum, 6(12%) and 5(10%) T.mentagrophytes and T.verrucosum and 4(8 %) isolates E. floccosum and T.erinacei and finally appeared 3(6 %) isolate T.schoenleinii and T.concentricum. The results of the statistical analysis showed non significant difference P &lt;0.05 in the total number of white blood cells and monocyte (5.15 ± 2.636, 0.89±0.51) in patients with dermatophytes compared with the group Control mean± SD (5.73±1.821, ± 0.69 ± 1.591) respectively. Lymphocytes significant decreased (1.82 ± 1.301) in patients with dermatophytes compared to control group (4.60 ± 1.35) P≤0.01. Furthermore granlocytes increased (2.30±1.591) in patients with dermatophytes compared to control group (0.33± 0.403) P&lt;0.01. The study concluded that perhaps the reduction of some of the body's WBCs parameters has an important role in the incidence of fungal infection in people infected with them when compared to healthy people.

Meta-atlas of Juvenile and Adult Enteric Neuron scRNA-seq for Dataset Comparisons and Consensus on Transcriptomic Definitions of Enteric Neuron Subtypes.

The enteric nervous system (ENS) is a complex network of interconnected ganglia within the gastrointestinal (GI) tract. Among its diverse functions, the ENS detects bowel luminal contents and coordinates the passing of stool. ENS defects predispose to GI motility disorders. Previously, distinct enteric neuron types were cataloged by dye-filling techniques, immunohistochemistry, retrograde labeling, and electrophysiology. Recent technical advances in single cell RNA-sequencing (scRNA-seq) have enabled transcriptional profiling of hundreds to millions of individual cells from the intestine. These data allow cell types to be resolved and compared to using their transcriptional profiles ("clusters") rather than relying on antibody labeling. As a result, greater diversity of enteric neuron types has been appreciated. Because each scRNA-seq study has relied on different methods for cell isolation and library generation, numbers of neuron clusters and cell types detected differs between analyses. Cell counts in each dataset are particularly important for characterization of rare cell types since small numbers of profiled cells may not sample rare cell types. Importantly, each dataset, depending on the isolation methods, may contain different proportions of cells that are not detected in other datasets. Aggregation of datasets can effectively increase the total number of cells being analyzed and can be helpful for confirming the presence of low-abundance neuron types that might be absent or observed infrequently in any single dataset. Here we briefly systematically review each Mus musculus single cell or single nucleus RNA-sequencing enteric nervous system dataset. We then reprocess and computationally integrate these select independent scRNA-seq enteric neuron datasets with the aim to identify new cell types, shared marker genes across juvenile to adult ages, dataset differences, and achieve some consensus on transcriptomic definitions of enteric neuronal subtypes. Data aggregation generates a consensus view of enteric neuron types and improves resolution of rare neuron classes. This meta-atlas offers a deeper understanding of enteric neuron diversity and may prove useful to investigators aiming to define alterations among enteric neurons in disease states. Future studies face the challenge of connecting these deep transcriptional profiles for enteric neurons with historical classification systems.

Yunpi Xiefei Huatan Tang decoction reduces airway inflammation and airway remodeling in asthmatic mice through Wnt/β-catenin signaling pathway.

Asthma is one of the most prevalent chronic respiratory diseases among children, markedly impairing patient's health and imposing an increasing burden on the healthcare system. Several traditional Chinese medicines have demonstrated efficacy in alleviating asthma symptoms through studies conducted on animal models. Recent studies have shown that the Yunpi Xiefei Huatan Tang decoction (YPD) exhibits significant therapeutic outcomes in treating phlegm-obstructed pulmonary asthma. However, the precise regulatory effects of YPD on the progression of asthma require additional investigation. To explore the functions of YPD in asthma progression. The asthma rat model triggered by ovalbumin (OVA) was established successfully. The pathological changes of lung tissues were examined through Hematoxylin and Eosin (H&E) staining. The levels of Interleukin 6 (IL-6) and IL-1β were tested through Enzyme-Linked-Immunosorbent Serologic Assay (ELISA). The number of total cells or eosinophils in bronchoalveolar lavage fluid was confirmed through cell counter. The collagen deposition in bronchi was assessed through Masson staining. The protein expressions were measured through western blot. This study demonstrated that YPD could mitigate airway inflammation in an OVA-triggered asthma rat model. Furthermore, YPD was found to decrease the production of inflammatory cytokines in the lungs and suppress the infiltration of inflammatory cells into bronchoalveolar lavage fluid. Additionally, the airway remodeling stimulated by OVA could be suppressed following YPD treatment. Finally, it was disclosed that YPD inhibited the wingless-related integration site-beta-catenin (Wnt/β-catenin) signaling pathway in the OVA-stimulated asthma rat model. YPD alleviated airway inflammation and remodeling in asthmatic mice via the Wnt/β-catenin signaling pathway. This research offers significant insights into the potential application of YPD in the treatment of asthma.

Water-avoidance stress aggravates prostatic inflammation in a murine model of chronic prostatitis.

To date, few studies have considered the influence of psychological factors on chronic prostatitis (PRO) models. Here, we aimed to refine a murine PRO model combining chemically induced prostatitis with psychological stress. A total of 40 mice were randomly divided into four groups: normal control (NC) group, PRO group, water avoidance stress (WAS) group, and PRO + WAS group. Ten mice were assigned to each group: five for cystometrograms (CMGs) and five for von Frey testing and histological analysis. PRO was induced through a prostatic injection of 10% paraformaldehyde. The WAS mice were placed on the middle platform for 1 h per day for 10 consecutive days. The results of the von Frey test demonstrated that both WAS and PRO induced bladder hyperalgesia in mice, and the WAS + PRO group showed significant pelvic pain symptoms either. The CMG results suggested that the PRO group, the WAS group, and the PRO + WAS group all exhibited bladder overactivity, presented as a shortened micturition interval and decreased threshold pressure evoking bladder contraction. The symptoms of the PRO group and the PRO + WAS group were more severe than those of the WAS group. The tissue staining results indicated that WAS itself caused only mild prostatic inflammation but could significantly aggravate chemical-induced prostatic inflammation, as well as the total number of mast cells and proportion of activated mast cells. Our refined murine PRO model could manifest persistent bladder overactivity, pelvic hyperalgesia and prostatic inflammation. WAS could induce mild prostatic inflammation and aggravate primary prostatic inflammation.

Assessment of using heart of date palm as a new source of protein and carbohydrate on the quality of low-fat bio fermented camel milk and its potential anticancer properties

The heart of date palm (HDP) is a nutrient-dense vegetable, rich in protein and carbohydrate, obtained from the core of palm trees. Carbohydrates are the major component of the heart of date palm, providing a quick source of energy and fiber content can help digestion and contribute to feeling of fullness, making it good addition to a balanced diet. The study investigated the potential of incorporating HDP into low-fat bio fermented camel milk (BCM). The initial findings highlighted HDP's medicinal properties and its significant cytotoxic effects against HepG2 cells, indicating its potential as an anticancer agent. Over 21 days of cold storage, the addition of HDP improved the water-holding capacity, color, sensory qualities, bioactive compounds, and rheological properties of low-fat BCM. Moreover, no mold, yeasts, or coliforms were detected in any samples throughout the storage. The total number of viable probiotic cells remained functional (>106 log CFU/g) for 21 days. The sensory evaluation results suggested that high-quality low-fat BCM can be achieved by adding up to 20 % HDP. The molecular docking study supports the various biological activities of HDP particularly in relation to its cytotoxic effects against HepG2 cancer cells. This study opens the door for further exploration into utilizing HDP as a novel functional ingredient in dairy foods to create value-added products.

Features of Brain Damage after Neutron Irradiation of the Head and Modification of the Damage by Lactoferrin.

: Mouse heads were irradiated in a beam of neutrons and gamma rays from the IR-8 nuclear reactor. The brain cells of control and irradiated mice were isolated using Percoll. Neurons and resting and activated microglial cells were analyzed using the fluorescently labeled antibodies and flow cytometry. The level of DNA double-strand breaks in neurons was determined by γH2AX histone content. Cytokine gene expression in the hippocampus was studied by RT-PCR. Behavior and cognitive functions were studied using the open field, Morris water maze, and novel object recognition tests. LF was isolated from female colostrum by preparative ion-exchange chromatography and purified by affinity chromatography on heparin-Sepharose. : γ,n-Irradiation of the mouse head at a dose of 1.5 Gy led to an increase in the level of DNA double-strand breaks in neurons. Twenty-four hours after irradiation the total number of cells and the number of neurons in the isolated fraction of brain cells decreased, but the number of microglial cells remained unchanged. The number of resting and activated microglia did not change within 3-72 h after γ,n-irradiation. The expression level of the TNFα, IL-1β, and IL-6 genes increased 2 months after γ,n-irradiation of the mouse head at a dose of 1.5 Gy, indicating the development of neuroinflammation. At this time, irradiated mice demonstrated the anxiety-like behavior and impaired spatial and episodic memory. A single i.p. administration of human LF to mice immediately after γ,n-irradiation of the head did not affect the observed radiation-induced disturbances, but decreased the gene expression levels of TNFα, IL-1β, and IL-6 pro-inflammatory cytokines and increased the gene expression level of TGFβ anti-inflammatory cytokine in the hippocampus 2 months after radiation exposure. The obtained results indicate a partial decrease in the level of hippocampal neuroinflammation of irradiated animals treated with LF. . γ,n-Irradiation of the mouse head at a dose of 1.5 Gy leads to DNA damage of neurons and the decrease in the number of neurons. Microglia cells are more resistant to such radiation exposure. Late after head-only γ,n-irradiation, mice develop neuroinflammation, which is detected by an increase in the pro-inflammatory cytokine gene expression in the hippocampus and also by anxiety-like behavior and impaired cognitive functions. A single LF administration leads to a partial decrease in the neuroinflammation level, but does not affect the other studied parameters. The optimal dosing regimen of LF remains to be determined to preserve cognitive functions after γ,n-irradiation of the brain.

Assessment of loliolide extracted from Biserula pelecinus, present during in vitro oocyte maturation, on fertilisation and embryo development in sheep

Context As a ‘duty of care’, it is important to test whether new forage plants for ruminants contain secondary compounds (PSCs) that affect reproductive performance. We have previously observed, a posteriori, that the presence of a methanolic extract of Biserrula pelecinus during maturation of sheep oocytes increased fertilisation rate and blastocyst development. This result needed to be verified a priori and, if the outcome was repeated, we needed to identify the plant secondary metabolite responsible. Aims To test whether PSCs from B. pelecinus, when added to the oocyte maturation medium, improve fertilisation rate and blastocyst development; to test whether loliolide is the active molecule produced by B. pelecinus. Methods Methanol–chloroform extracts of B. pelecinus were fractionated using rapid silica filtration and solvents of increasing polarity. Fractions at final concentrations of 0, 100 or 200 μg mL−1 were added to the medium used to mature sheep cumulus–oocyte complexes (COCs) and effects were determined for maturation, subsequent cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency and total blastocyst cell number (TCN). Results Fraction BP-6 at 100 μg mL−1 reduced blastocyst rate (P &lt; 0.05), but had no effect when the dose was doubled to 200 μg mL−1. Further fractionation using semi-preparative high-performance liquid chromatography showed loliolide as the most abundant compound in BP-6. Supplementation of the in vitro maturation medium with loliolide (0, 2.5, 5, 10 and 25 μg mL−1) did not affect any measure of embryo development. All COCs treated with B. pelecinus fractions reached the final stage of embryo development, blastocyst hatching. Total blastocyst cell number was not affected. Conclusion The presence of fractions of B. pelecinus extract during in vitro oocyte maturation can reduce embryo development. Implications In vitro techniques can detect potential effects of forages on reproduction. Some fractions from an extract of B. pelecinus when present during oocyte maturation can reduce embryo development. The abundant PSC, loliolide, was not responsible. There was no indication that a PSC in B. pelecinus improves outcomes.

Enhanced Fear Extinction Through Infralimbic Perineuronal Net Digestion: The Modulatory Role of Adolescent Alcohol Exposure.

Perineuronal nets (PNNs) are specialized components of the extracellular matrix that play a critical role in learning and memory. In a Pavlovian fear conditioning paradigm, degradation of PNNs affects the formation and storage of fear memories. This study examined the impact of adolescent intermittent ethanol (AIE) exposure by vapor inhalation on the expression of PNNs in the adult rat prelimbic (PrL) and infralimbic (IfL) subregions of the medial prefrontal cortex. Results indicated that following AIE, the total number of PNN positive cells in the PrL cortex increased in layer II/III but did not change in layer V. Conversely, in the IfL cortex, the number of PNN positive cells decreased in layer V, with no change in layer II/III. In addition, the intensity of PNN staining was significantly altered by AIE exposure, which narrowed the distribution of signal intensity, reducing the number of high and low intensity PNNs. Given these changes in PNNs, the next experiment assessed the effects of AIE and PNN digestion on extinction of a conditioned fear memory. In Air control rats, digestion of PNNs by bilateral infusion of Chondroitinase ABC (ChABC) into the IfL cortex enhanced fear extinction and reduced contextual fear renewal. In contrast, both fear extinction learning and contextual fear renewal remained unchanged following PNN digestion in AIE exposed rats. These results highlight the sensitivity of prefrontal PNNs to adolescent alcohol exposure and suggest that ChABC-induced plasticity is reduced in the IfL cortex following AIE exposure.

Oviduct epithelial spheroids during in vitro culture of bovine embryos mitigate oxidative stress, improve blastocyst quality and change the embryonic transcriptome

BackgroundIn vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8–16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen levels (5% vs. 20%).MethodsBovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7–8. Expanded blastocysts on days 7–8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing.ResultsUnder 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls (P < 0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture (P < 0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to external signals, early embryo development, and transport of small molecules among the most significantly impacted.ConclusionOES had beneficial effects on embryo development and quality under both 5% and 20% oxygen, mitigating oxidative stress. Stronger effects on embryo quality and transcriptome were obtained after 7 than 4 days of co-culture. This study shows the impact of OES on embryo development and reveals potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development.

The Effect of Simulated Physiological Oocyte Maturation (SPOM) and L-Carnitine on Bovine Oocyte Developmental Competence.

Simulated Physiological Oocyte Maturation (SPOM) mimics in vitro the physiological events of oocyte maturation in the presence of cAMP modulators. These modulators increase the intracellular concentrations of cAMP, which inhibits the immediate resumption of meiosis and gives the oocyte more time to gain optimal developmental competence. In addition, L-carnitine helps to increase the energy supply of cells through the β-oxidation of fatty acids. This study aimed to investigate the effect of SPOM and L-carnitine supplementation during In Vitro Maturation (IVM) and In Vitro Culture (IVC) on the developmental competence of bovine oocytes. Ovarian Cumulus Complexes (COCs) were cultured in the presence or absence of forskolin+IBMX during the first 2 hr of IVM (pre-IVM) with or without L-carnitine (LC) during IVM or IVC in six experimental groups as follows: I) pre-IVM (pre-IVM group), II) pre-IVM with L-carnitine supplementation during IVM (pre-IVM/LC group), III) L-carnitine supplementation during IVM (IVM/LC group), IV) L-carnitine supplementation during in vitro culture (IVC/LC group), V) pre-IVM+ IVC/LC group, and VI) no treatment during IVM and IVC (Control group). The cleavage and blastocyst rates, the blastocysts' total cells number, and the expression of Nanog, Bax, Oct4, Cdx2, and Ifnt genes in resulting blastocysts were assessed. To assess differences among experimental groups, a one-way analysis of variance was initially employed, followed by post hoc Fisher LSD. The difference between groups was considered statistically significant when p<0.05. The cleavage and blastocyst rates in the Pre-IVM and Pre-IVM/LC groups was higher than control group and other groups (p≤0.05) except for IVC/LC and IVM/LC groups, respectively. The number of blastocyst's Inner Cell Mass (ICM) in pre-IVM and Pre-IVM/LC groups as well as the ratio of ICM/TE were higher than control group (p<0.05). The expression of OCT4, CDX2, and IFNT increased in both the pre-IVM and pre-IVM/LC groups compared to the control group (p<0.05). In conclusion, the application of SPOM-adapted IVM and L-carnitine during IVM of bovine oocyte improves the quantity and quality of the resulting embryos.

Blood MAIT cells phenotype in patients with Opisthorchis felineus invasion depending on the severity of liver fibrosis

Introduction. MAIT cells are a new subpopulation of T cells that protect mucous barriers against penetration of foreign substances. There are practically no studies devoted to the participation of these cells in the pathogenesis of parasitic diseases.Aim. To study the phenotype of blood MAIT cells in patients with Opisthorchis felineus (O. felineus) invasion depending on the severity of liver fibrosis.Materials and methods. A total of 78 patients with O. felineus invasion (42 men and 36 women) and 26 control group individuals (14 men and 12 women) were examined. Opisthorchiasis was diagnosed using coproovoscopy and duodenal contents microscopy. All patients underwent liver elastometry using Aixplorer (France) or Siemens Acuson S2000 (Germany) systems with determination of the liver fibrosis degree according to METAVIR. The phenotype composition of lymphocytes was investigated using a Navios flow cytometer (Beckman Coulter, USA). T cells, T helpers, and T cytotoxic lymphocytes were isolated and the presence of NCR Va7.2 and CD161 on the surface of these cells was assessed.Results. The content of MAIT T-helpers was decreased in patients with O. felineus invasion compared to healthy individuals (p &lt; 0.001). In MAIT T-cytotoxic cells, a similar pattern was not detected (p = 0.5). In patients with liver fibrosis F2 according to METAVIR compared to individuals with F0 according to METAVIR, a decrease in the total number of T-cells, T-helpers and T-cytotoxic cells, as well as MAIT T-helpers and MAIT T-cytotoxic cells was observed. Thus, the content of CD161+ NCR Va7.2+ T-helpers was 0.020% [0.004–0.042%] in patients with opisthorchiasis with F0 according to METAVIR and 0.0% [0.0–0.003%] in individuals with liver fibrosis F2 according to METAVIR (p = 0.001). For CD161+ NCR Va7.2+ T-cytotoxic cells, these indicators were, respectively, 1.47% [0.41–2.49%] and 0.12% [0.07–0.31%] (p &lt; 0.001).Conclusion. Further study of MAIT cells in patients with liver pathology has undoubted prospects for the creation of new therapeutic and diagnostic technologies.

P53 ABNORMAL ENDOMETRIAL CANCERS – ANALYSIS OF PD-1 CO-EXPRESSION BY CD4 HELPER AND CD8 CYTOTOXIC T-CELL SUBTYPES IN THE TUMOR MICROENVIRONMENT

Abstract Introduction/Objective Endometrial cancer is classified into four molecular subtypes of which the p53 abnormal variant is biologically aggressive. Programmed cell death protein 1 (PD-1) is a cell surface receptor and a vital inhibitor of the immune response. Programmed death ligand 1 (PD-L1) is a trans-membrane co-inhibitory factor of the immune response. PD-1 / PDL-1 interaction inhibits the activation and survival of T cells and decreases cytokine secretion. Some cancer cells express PD-L1, which then bind to PD-1 expressed on tumor specific T cells, and escape destruction by the T cells. The CD4/CD8 T cell immune response in the p53 abnormal variant has not been adequately evaluated with respect to the PD-1/PD-L1 pathway. Understanding this immune microenvironment is important to assess the potential use of anti-PD-1 targeted tumor therapy. Methods/Case Report Twenty cases of p53 abnormal endometrial cancers were obtained. All cases were PDL-1 +ve by immunohistochemistry. Two dual stains were performed - PD-1/CD4 and PD-1/CD8 antibodies. Six high-power fields (40x objective) with the greatest concentration of lymphocytes were counted. The total number of CD4 or CD8-only cells was recorded for each of the two stained slides for each case. The total number of PD- 1/CD4 and PD-1/CD8 positive cells were counted and recorded. The results were tabulated, and a paired t-test statistical analysis was performed to compare the total number of CD4 and CD8 cells and the percentage of PD-1/CD4 cells with the percentage of PD-1/CD8 positive cells. Results (if a Case Study enter NA) A statistically significant increase in the number of CD8 (Mean = 383) compared to CD4 cells (Mean = 317) was seen in the tumor microenvironment (p-value=0.0023). Evaluating PD-1 expression, the converse was true, and the percentage of PD-1/CD4 cells (Mean = 13.035%) was increased compared to the percentage of PD-1/CD8 cells (Mean = 9.205%), achieving statistical significance (p-value=0.0116). Conclusion The statistically significant increase in CD8 cells in the tumor and its stroma suggests that a robust cytotoxic response is being mounted to this high-risk molecular variant. The concurrent increase in CD4/PD-1 positive lymphocytes provides an appropriate microenvironment for the use of anti-PD-1 immunotherapy, since blockade of PD-1 on CD4 helper T cells may augment the tumor ablative cytotoxic properties of the CD8 positive lymphocytes. This includes reinvigoration of exhausted CD8+ T cells. Anti-PD-1 may also prevent the inhibition of PD-1 positive CD8 cells.

Mitoquinone improves porcine embryo development through modulating oxidative stress and mitochondrial function

Oxidative stress caused by excess reactive oxygen species (ROS) is one of the main causes of low efficiency in in vitro production of embryos. These ROS can cause mitochondrial dysfunction and apoptosis, resulting in poor embryo development. Therefore, to prevent mitochondrial damage and apoptosis caused by ROS, we investigated the effects of mitoquinone (MitoQ), a mitochondrial-targeted antioxidant, on the in vitro culture (IVC) of porcine embryos. Various concentrations of MitoQ (0, 0.01, 0.1, or 1 nM) were supplemented during the entire period of IVC. The results showed that supplementation with 0.1 nM MitoQ significantly increased the blastocyst formation rate, with a higher total cell number including trophectoderm cell number and higher transcript expression of lineage-specific transcription factors in blastocysts. In addition, the 0.1 nM MitoQ-treated group showed a significantly lower percentage and number of apoptotic cells in blastocysts with positively regulated transcript expression of apoptosis-related genes. Therefore, 0.1 nM MitoQ was suggested as optimal concentration for porcine IVC and used for further investigations. MitoQ treatment significantly reduced intracellular ROS levels and increased glutathione levels in Day 2 embryos, with upregulated the transcript expression of antioxidant enzymes-related genes. Furthermore, the MitoQ group exhibited a significantly higher mitochondrial quantity, mitochondrial membrane potential, and ATP content in Day 2 embryos, with increased transcript expression of mitochondrial biogenesis-related genes. Taken together, these findings reveal that MitoQ supplementation can enhance the developmental competence of porcine embryos by decreasing oxidative stress and improving mitochondrial function.

8235 Mutations in EMX2, a Homeodomain Transcription Factor, Cause Idiopathic Hypogonadotropic Hypogonadism

Abstract Disclosure: M. Stamou: None. M. Tompkin: None. H. Bow: None. H. Brand: None. L. Plummer: None. M. Talkowski: None. Y. Shen: None. D. Wu: None. R. Balasubramanian: None. S. Wray: None. S.B. Seminara: None. The genetic etiology of infertility remains largely unknown. To identify novel human infertility genes, we applied a de novo rare variant analysis in 142 parent-proband trios with idiopathic hypogonadotropic hypogonadism (IHH), a Mendelian disease caused by Gonadotropin Releasing Hormone (GnRH) deficiency. The discovery pipeline identified 43 rare de novo copy number variants (CNVs-deletions) spanning 263 genes and 213 rare de novo single nucleotide variants (SNVs) in 191 genes. Four genes were found to be affected by both rare de novo CNVs and SNVs among unrelated sporadic IHH probands. A rare variant association testing for protein truncating variants (PTVs) in the IHH cohort (N=1,394) compared to gnomAD controls (N=125,784) revealed that 3/4 genes demonstrated enrichment for PTVs in the IHH cohort compared to controls: the 2 known for IHH genes (ANOS1 and FGFR1) and one novel candidate gene, EMX2. IHH probands with de novo EMX2 variants demonstrated developmental delay and hearing loss. Common EMX2 variants were linked to delayed puberty/infertility, hearing loss and Parkinson’s disease in a phenome-wide association study of the Massachusetts General Brigham Biobank (N=65,253). Emx2 lineage tracing was conducted on Emx2Cre/+; RosatdT/+ and Emx2Cre/-; RosatdT/+ mouse embryos at embryonic day(E) 13.5. The tdT reporter was co-expressed in neuroendocrine GnRH (nGnRH) cells as they migrated from the olfactory pit to the nasal forebrain junction. WT and KO embryos from Emx2+/- matings were immunostained for GnRH across E12.5, E13.5, E14.5 and E16. The total number of nGnRH cells was greater in the Emx2 KO mice compared to WT littermates. However, by E16.5, fewer nGnRH cells were detected in the forebrain of Emx2 KO, compared to WT (WT= 496±28, KO=384±147, p=0.0125). Thus, failure of nGnRH neurons to enter the forebrain could contribute to the IHH pathogenesis. In conclusion, by utilizing a de novo variant analysis and a mouse model, EMX2 was uncovered as a novel gene for human infertility. Presentation: 6/1/2024