Basal Levels Research Articles

P-803 Intra-ovarian injection of plasma rich in stem cell-secreted and platelet-enclosed factors to promote follicle rescue and reproductive outcomes in women with very low ovarian reserve

Abstract Study question Could intraovarian injection of plasma rich in stem-cell and platelet growth factors optimize ovarian reserve and IVF outcomes in women with very low ovarian reserve? Summary answer Intraovarian injection of G-CSF mobilized plasma rich in grown factors improved follicle count since first follow-up and led to better fecundation, implantation and pregnancy’ rates. What is known already Autologous stem cell ovarian transplantation (ASCOT) of bone marrow derived stem cells optimized ovarian reserve and reproductive outcomes in poor responders (POR) and this effect seems to be due to the growth factors secreted by the stem cells, acting through paracrine pathways. Similarly, platelet-rich Plasma (PRP) has been also offered to promote ovarian reactivation in patients with impaired ovarian reserve by providing positive growth factors contained in platelets. Indeed, a technique that combines both the non-cellular components of ASCOT and PRP (ASCOT-1) has been proposed to optimize follicle rescue and IVF outcomes in women with very low reproductive prognosis. Study design, size, duration Retrospective observational study with 159 women (≤ 45 years) diagnosed with POR and primary ovarian insufficiency (POI) according to ESHRE criteria, who underwent the ASCOT-1 technique after refusing oocyte donation, between June 2021-October 2023 at IVIRMA Alicante, Spain (IRB approval 2310-FIVI-188-SH). The ASCOT-1 consisted in the intraovarian injection of plasma rich in both stem cell-secreted and platelet-enclosed factors obtained after granulocyte colony stimulating factor (G-CSF) mobilization and PRP obtention (Endoret Kit BTI, Biotechnology institute). Participants/materials, setting, methods Medical records of patients who underwent the ASCOT-1 as an ovarian reactivation treatment were examined. Follow up of AFC and AMH was carried out for a maximum of 4 menstrual cycles to evaluate follicular activation. Moreover, when follicle growth was detected, patients underwent a controlled ovarian stimulation (COS). IVF and reproductive outcomes were compared with previous COS cycles started in our clinics, when available. T-test was used where appropriate. Main results and the role of chance Overall 159 POR/POI women (age 38.7±4.2years, AMH0 0.22±0.41ng/ml; AFC0 1.4±1.9) underwent the ASCOT-1 technique. Mean volume of plasma injected was 6.8±0.8ml, distributed equally in both ovaries. First follow-up was done in 131 women showing statistically significant improvements in the AFC during the entire follow-up compared to the basal levels measured before the treatment (AFC1 3.3±3.4, p < 0.001; AFC2 3.3±3.6, p < 0.001; AFC3 2.9±2.6, p < 0.001; AFC4 2.4±2.6, p = 0.07). After ASCOT-1, 98 COS were initiated in 48 women in our clinics, which were compared to their previous 66 cycles. Although age was significantly higher in their posterior cycles (38.0±2.9 vs40.0±3.6; p < 0.001), after ASCOT-1 oocyte retrieval was reached in a greater amount of cases (75.5% vs87.5%, p=NS). ASCOT-1 showed a positive, but non statistically significant effect in the number of MII-oocytes (2.8±4.1 vs3.2±3.9, p=NS) and blastocysts obtained (1.8±2.8 vs2.4±3.1, p=NS), but relevant improvements were observed in quality related parameters such as oocyte maturation (61.5%, 95%IC 45.8-77.3, vs86.1%, 95I%IC 69.9-85.5; p 0.03), fertilization (62.6%, 95%CI 53.0-71.3, vs76.5%, 95%CI 70.0-82.0; p < 0.001), implantation (25.0%, 95%CI 8.3-52.5 vs41.1%, 95%CI 19.4-66.5; p < 0.001) and clinical pregnancy rate (25.0%, 95%CI 8.3-52.5 vs46.6%, 95%CI 22.2-72.5; p < 0.001). Ultimately, 14 clinical pregnancies (7 after ET and 7 by natural conception) were obtained after ASCOT-1. Limitations, reasons for caution This retrospective study involves short periods of follow up and limited amount of IVF attempts. Thus, further prospective studies with increased sample size are required to validate our results, to establish the duration of the effects, and to identify which subgroups of patients could benefit the most from this treatment. Wider implications of the findings Growth factors contained in the G-CSF mobilized PRP could play a role in follicular development, providing a new tool to increase the reproductive potential of women with very low ovarian reserve where oocyte donation is the only practical option, allowing them to access to homologous IVF treatment. Trial registration number 2310-FIVI-188-SH

O-197 Correlation of intracellular pH of non-normozoospermic men with fertilization rates in assisted reproduction procedures (Pilot study)

Abstract Study question Does measuring qualitative changes in the sperm pHi could predict the fertilizing potential of a non-normozoospermic patients under fertility treatment? Summary answer The increase of pHi correlates with fertilization rates. What is known already The intracellular pHi (pHi) is of great importance for a wide variety of sperm physiological processes, including its motility. The pHi regulates several key sperm proteins, such as the Ca2+ channel, CatSper (sperm cation channel) which is involved in sperm hyperactivation and the K+ channel SLO3.The latter participates in the regulation of membrane potential during capacitation. Interestingly, human sperm pHi has been found to positively correlate with both hyperactivated motility and in vitro fertilization (IVF) success in normozoospermic patients. Study design, size, duration This prospective study employed unidentified semen samples from 62 non-normozoospermic men (patients, age 38 ± 4 years old) undergoing fertility treatment from January to November 2023 at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and procedures with less than 4 oocytes were excluded. The pHi of sperm samples was evaluated by time-lapse flow cytometry. For comparison, the pHi of 13 normozoospermic men (donors, age 28 + 6 years old) were also analyzed. Participants/materials, setting, methods Sperm were separated by density gradient and incubated in capacitating HTF media to be used for conventional IVF or ICSI. Cells were stained with vitality (propidium iodide) and pHi (BCECF-AM) sensitive fluorescent dyes. Fluorescence changes were evaluated using an AccuriC6 flow cytometer. For each pHi recording, 10 mM NH4Cl were used as an alkalinization positive control and for data normalization. Basal levels of sperm pHi and its correlation with fertilization rates, were performed using R. Main results and the role of chance The changes in pHi were recorded as changes in the BCECF fluorescence. The increase in fluorescence is proportional to an increase in pHi and vice versa. Interestingly, we observed that the increase in pHi in sperm used for ICSI but not for IVF procedures, which were paired with oocytes from female donors, are positively and significantly correlated with higher fertilization rates (R = 0.68; *p=0.02). This seems to be the same for ICSI treatment pairing sperm with oocyte from the female partner, although the correlation is not yet statistically significant (R = 0.44; p = 0.12). We did not find significant differences in the basal pHi values between sperm from donors and patients; however, we observed that sperm from patients, had higher pHi values (0.57 + 0.15 a.u.f) compared to sperm from donors (0.48 + 0.24 a.u.f). Our results suggest that sperm pHi may be an important regulator for the process of fusion and not only for fertilization. Qualitative sperm pHi values can contribute to predicting fertilization success in patients undergoing ICSI treatment. Limitations, reasons for caution The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples. Physiological responses may vary even among the same patient or cell’s donor. Our population-based analysis could be underestimating such single cell variations. Wider implications of the findings Current protocols for sperm analysis fail to successfully predict the fertilizing capacity of semen samplest.Measuring pHi in capacitated sperm, could be a promising tool to help clinicians choose the best fertilization technique for each couple. This protocol is easy to perform in a laboratory that has a flow cytometer. Trial registration number Not applicable

Increased Urine Excretion of Neutrophil Granule Cargo in Active Proliferative Lupus Nephritis

Background: Lupus nephritis (LN) occurs in over half of patients with systemic lupus erythematosus (SLE), but the cellular and molecular events that contribute to LN are not clearly defined. We reported previously that neutrophil degranulation participates in glomerular injury in mouse models of acute LN. The current study tests the hypothesis that glomerular recruitment and subsequent activation of neutrophils results in urine excretion of neutrophil granule constituents that are predictive of glomerular inflammation in proliferative LN. Methods: Urine and serum levels of 11 neutrophil granule proteins were measured by antibody-based array in proliferative LN patients and healthy donors (HD), and results were confirmed by ELISA. Glomerular neutrophil accumulation was assessed in LN patient biopsies who contributed urine for granule cargo quantitation and normal kidney tissue by microscopy. Degranulation was measured by flow cytometry in neutrophils isolated from LN patients and HD controls by cell surface granule markers CD63 (azurophilic), CC66b (specific) and CD35 (secretory). Nonparametric statistical analyses were performed and corrected for multiple comparisons. Results: Eight granule proteins (myeloperoxidase, neutrophil elastase, azurocidin, olfactomedin-4, lactoferrin, alpha-1-acid glycoprotein 1 (α-1AG), MMP-9, and cathelicidin) were significantly elevated in urine from patients with active proliferative LN by array and/or ELISA, while only neutrophil elastase was increased in LN serum. Urine excretion of α-1AG declined in patients who achieved remission. The majority of LN glomeruli contained ≥ 3 neutrophils. Basal levels of specific granule markers were increased in neutrophils from LN patients, compared to HD controls. Serum from patients with active LN stimulated specific and secretory, but not azurophilic granule release by HD neutrophils. Conclusions: Circulating neutrophils in patients with LN are primed for enhanced degranulation. Glomerular recruitment of those primed neutrophils leads to release and urine excretion of neutrophil granule cargo that serves as a urine marker of active glomerular inflammation in proliferative LN.

Atypical Course of the Habenulo-Interpeduncular Tract in Chick Embryos.

Classical studies of the avian diencephalon hardly mention the habenulo-interpeduncular tract (a.k.a. retroflex tract), although both the habenula (HB) (its origin) and the interpeduncular nuclear complex (its target) are present. Retroflex tract fibers were described at early embryonic stages but seem absent in the adult in routine stains. However, this tract is a salient diencephalic landmark in all other vertebrate lineages. It typically emerges out of the caudal HB, courses dorsoventrally across thalamic alar and basal plates just in front of the thalamo-pretectal boundary, and then sharply bends 90° caudalwards at paramedian basal plate levels (this is the "retroflexion"), to approach longitudinally via paramedian pretectum and midbrain the rostralmost hindbrain, specifically the prepontine median interpeduncular complex across isthmus and rhombomere 1. We systematize this habenulo-interpeduncular course into four parts named subhabenular, retrothalamic, tegmental, and interpeduncular. We reexamined the chicken habenulo-interpeduncular fibers at stages HH30 and HH35 (6.5- and 9-day incubation) by mapping them specifically with immunoreaction for BEN protein, a well-known marker. We found that only a small fraction of the stained retroflex tract fibers approaches the basal plate by coursing along the standard dorsoventral pathway in front of the thalamo-pretectal boundary. Many other habenular fibers instead diverge into atypical dispersed courses across the thalamic cell mass (implying alteration of the first subhabenular part of the standard course) before reaching the basal plate; this dispersion explains their invisibility. A significant number of such transthalamic habenular fibers cross orthogonally the zona limitans (ZLI) (the rostral thalamic boundary) and invade the caudal alar prethalamus. Here, they immediately descend dorsoventrally, just rostrally to the ZLI, until reaching the prethalamic basal plate, where they bend (retroflex) caudalwards, entering the thalamic basal paramedian area. These atypical fibers gradually fasciculate with the other groups of habenular efferent fibers in their final longitudinal approach to the hindbrain interpeduncular complex. We conclude that the poor visibility of this tract in birds is due to its dispersion into a diversity of atypical alternative routes, though all components eventually reach the interpeduncular complex. This case merits further analysis of the diverse permissive versus nonpermissive guidance mechanisms called into action, which partially correlate distinctly with successive diencephalic, mesencephalic, and hindbrain neuromeric fields and their boundaries.

Carryover effects of embryonic hypoxia exposure on adult fitness of the Pacific abalone

The widespread and severe drop in dissolved oxygen concentration in the open ocean and coastal waters has attracted much attention, but assessments of the impacts of environmental hypoxia on aquatic organisms have focused primarily on responses to current exposure. Past stress exposure might also affect the performance of aquatic organisms through carryover effects, and whether these effects scale from positive to negative based on exposure degree is unknown. We investigated the carryover effects of varying embryonic hypoxia levels (mediate hypoxia: 3.0−3.1 mg O2/L; severe hypoxia: 2.0−2.1 mg O2/L) on the fitness traits of adult Pacific abalone (Haliotis discus hannai), including growth, hypoxia tolerance, oxygen consumption, ammonia excretion rate, and biochemical responses to acute hypoxia. Moderate embryonic hypoxia exposure significantly improved the hypoxia tolerance of adult Pacific abalone without sacrificing growth and survival. Adult abalone exposed to embryonic hypoxia exhibited physiological plasticity, including decreased oxygen consumption rates under environmental stress, increased basal methylation levels, and a more active response to acute hypoxia, which might support their higher hypoxia tolerance. Thus, moderate oxygen declines in early life have persistent effects on the fitness of abalone even two years later, further affecting population dynamics. The results suggested that incorporating the carryover effects of embryonic hypoxia exposure into genetic breeding programs would be an important step toward rapidly improving the hypoxia tolerance of aquatic animals. The study also inspires the protection of endangered wild animals and other vulnerable species under global climate change.

Prestimulation parameters predicting the pregnancy outcomes after IVF-ET in patients with unexplained infertility.

Assisted Reproductive Technologies (ART) are considered to be the most effective treatment option for unexplained infertility. This study aims to investigate the pregnancy outcomes of women who received in-vitro fertilization-embryo transfer (IVF-ET) treatment for unexplained infertility and the contributing factors affecting these outcomes. The present study included 789 consecutive women with unexplained infertility who were treated with IVF-ET at the ART Clinic of the Health Sciences University, Ankara Etlik Zübeyde Hanım Gynecology Training and Research Hospital between January 2007 and December 2019. The contributing factors affecting these outcomes, such as body mass index (BMI), basal follicle-stimulating hormone (FSH), and antimullerian hormone (AMH), were evaluated retrospectively. Clinical pregnancy per cycle was 19.8% among patients recruited. No statistically significant difference was detected in terms of age, infertility duration, and BMI of the patients who achieved pregnancy and who failed to get pregnant after IVF-ET treatment. The basal FSH level was found to be significantly lower (p=0.001), and the AMH level was significantly higher in patients who had clinical pregnancy (p=0.001). The basal AMH cut-off value was calculated to be 3.34 ng/mL, and the basal FSH cut-off value was calculated as 7.26 IU/L for the prediction of clinical pregnancy. IVF-ET treatment can be applied as a successful treatment option in unexplained infertility cases. Although the basal FSH and AMH values ​are not the cut-off values that have high sensitivity and specificity, they are considered to be associated with pregnancy rates.

Visualizing lysosomes hypochlorous acid in Parkinson's disease models by a novel fluorescent probe

Heightened oxidative stress is the principal driver behind the altered metabolism of neurotransmitters within the brains of Parkinson's disease (PD). Hypochlorous acid (HClO), a variant of reactive oxygen species (ROS), plays a crucial role in several lysosomal activities. An irregular concentration of HClO may result in significant molecular damage and contribute to the onset of neurodegenerative disorders. Despite this, the precise role of lysosomal HClO in PD remains unclear, due to its fast reactivity and low levels. This is further complicated by the lack of effective in situ imaging techniques for accurately tracking its dynamics. Therefore, it is of great significance to use effective tools to map the lysosomal HClO during the pathological process of PD. In this study, we propose a fluorogenic probe named Lys-PTZ-HClO for the specific and sensitive detection of HClO. Lys-PTZ-HClO exhibits features like a fast response time (10 s) and a low detection limit (0.72 μM). Benefiting from its superior properties, the probe was used to visualize the basal HClO levels, and the variation of HClO levels in lysosomal of living cells. More importantly, this probe was successfully applied for the first time to reveal increased lysosomal HClO in a cellular model of PD.

Systematic analysis of the Candida albicans kinome reveals environmentally contingent protein kinase-mediated regulation of filamentation and biofilm formation in vitro and in vivo.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

Acetyl-CoA metabolism maintains histone acetylation for syncytialization of human placental trophoblast stem cells.

During pregnancy, placental-fetal nutrient allocation is crucial for fetal and maternal health. However, the regulatory mechanisms for nutrient metabolism and allocation in placental trophoblasts have remained unclear. Here, we used human first-trimester placenta samples and human trophoblast stem cells (hTSCs) to discover that glucose metabolism is highly active in hTSCs and cytotrophoblasts, but during syncytialization, it decreases to basal levels, remaining necessary for fueling acetyl-CoA and differentiation potential. Acetate supplementation could rescue syncytiotrophoblast fusion from glycolysis deficiency by replenishing acetyl-CoA and maintaining histone acetylation, thus rescuing the activation of syncytialization genes. Even brief glycolysis deficiency could permanently inhibit differentiation potential and promote inflammation, which could also be permanently rescued by brief acetate supplementation invivo. These results suggest that hTSCs retain only basal glycolytic acetyl-CoA metabolism during syncytialization to regulate cell fates via nutrient-responsive histone acetylation, with implications for our understanding of the balance between placental and fetal nutrition.

Selective Assembly of TRPC Channels in the Rat Retina during Photoreceptor Degeneration.

Transient receptor potential canonical (TRPC) channels are calcium channels with diverse expression profiles and physiological implications in the retina. Neurons and glial cells of rat retinas with photoreceptor degeneration caused by retinitis pigmentosa (RP) exhibit basal calcium levels that are above those detected in healthy retinas. Inner retinal cells are the last to degenerate and are responsible for maintaining the activity of the visual cortex, even after complete loss of photoreceptors. We considered the possibility that TRPC1 and TRPC5 channels might be associated with both the high calcium levels and the delay in inner retinal degeneration. TRPC1 is known to mediate protective effects in neurodegenerative processes while TRPC5 promotes cell death. In order to comprehend the implications of these channels in RP, the co-localization and subsequent physical interaction between TRPC1 and TRPC5 in healthy retina (Sprague-Dawley rats) and degenerating (P23H-1, a model of RP) retina were detected by immunofluorescence and proximity ligation assays. There was an overlapping signal in the innermost retina of all animals where TRPC1 and TRPC5 physically interacted. This interaction increased significantly as photoreceptor loss progressed. Both channels function as TRPC1/5 heteromers in the healthy and damaged retina, with a marked function of TRPC1 in response to retinal degenerative mechanisms. Furthermore, our findings support that TRPC5 channels also function in partnership with STIM1 in Müller and retinal ganglion cells. These results suggest that an increase in TRPC1/5 heteromers may contribute to the slowing of the degeneration of the inner retina during the outer retinal degeneration.

Ultrasound-Mediated Lysozyme Microbubbles Targeting NOX4 Knockdown Alleviate Cisplatin-Exposed Cochlear Hair Cell Ototoxicity.

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.

Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A.

Osteocytes are the primary mechano-sensitive cell type in bone. Mechanical loading is sensed across the dendritic projections of osteocytes leading to transient reductions in focal adhesion kinase (FAK) activity. Knowledge regarding the signaling pathways downstream of FAK in osteocytes is incomplete. We performed tyrosine-focused phospho-proteomic profiling in osteocyte-like Ocy454 cells to identify FAK substrates. Gsα, parathyroid hormone receptor (PTH1R), and phosphodiesterase 8A (PDE8A), all proteins associated with cAMP signaling, were found as potential FAK targets based on their reduced tyrosine phosphorylation in both FAK- deficient or FAK inhibitor treated cells. Real time monitoring of intracellular cAMP levels revealed that FAK pharmacologic inhibition or gene deletion increased basal and GPCR ligand-stimulated cAMP levels and downstream phosphorylation of protein kinase A substrates. Mutating FAK phospho-acceptor sites in Gsα and PTH1R had no effect on PTH- or FAK inhibitor-stimulated cAMP levels. Since FAK inhibitor treatment augmented cAMP levels even in the presence of forskolin, we focused on potential FAK substrates downstream of cAMP generation. Indeed, PDE8A inhibition mimicked FAK inhibition at the level of increased cAMP, PKA activity, and expression of cAMP-regulated target genes. In vitro kinase assay showed that PDE8A is directly phosphorylated by FAK while immunoprecipitation assays revealed intracellular association between FAK and PDE8A. Thus, FAK inhibition in osteocytes acts synergistically with signals that activate adenylate cyclase to increase intracellular cAMP. Mechanically-regulated FAK can modulate intracellular cAMP levels via effects on PDE8A. These data suggest a novel signal transduction mechanism that mediates crosstalk between mechanical and cAMP-linked hormonal signaling in osteocytes.

Initial decrease in the lipoprotein(a) level is a novel prognostic biomarker in patients with acute coronary syndrome.

Lipoprotein(a) [Lp(a)] is a causal risk factor for atherosclerotic cardiovascular diseases; however, its role in acute coronary syndrome (ACS) remains unclear. To investigate the hypothesis that the Lp(a) levels are altered by various conditions during the acute phase of ACS, resulting in subsequent cardiovascular events. From September 2009 to May 2016, 377 patients with ACS who underwent emergent coronary angiography, and 249 who completed ≥ 1000 d of follow-up were enrolled. Lp(a) levels were measured using an isoform-independent assay at each time point from before percutaneous coronary intervention (PCI) to 48 h after PCI. The primary endpoint was the occurrence of major adverse cardiac events (MACE; cardiac death, other vascular death, ACS, and non-cardiac vascular events). The mean circulating Lp(a) level decreased significantly from pre-PCI (0 h) to 12 h after (19.0 mg/dL to 17.8 mg/dL, P < 0.001), and then increased significantly up to 48 h after (19.3 mg/dL, P < 0.001). The changes from 0 to 12 h [Lp(a)Δ0-12] significantly correlated with the basal levels of creatinine [Spearman's rank correlation coefficient (SRCC): -0.181, P < 0.01] and Lp(a) (SRCC: -0.306, P < 0.05). Among the tertiles classified according to Lp(a)Δ0-12, MACE was significantly more frequent in the lowest Lp(a)Δ0-12 group than in the remaining two tertile groups (66.2% vs 53.6%, P = 0.034). A multivariate analysis revealed that Lp(a)Δ0-12 [hazard ratio (HR): 0.96, 95% confidence interval (95%CI): 0.92-0.99] and basal creatinine (HR: 1.13, 95%CI: 1.05-1.22) were independent determinants of subsequent MACE. Circulating Lp(a) levels in patients with ACS decreased significantly after emergent PCI, and a greater decrease was independently associated with a worse prognosis.

Pro-Oxidant Auranofin and Glutathione-Depleting Combination Unveils Synergistic Lethality in Glioblastoma Cells with Aberrant Epidermal Growth Factor Receptor Expression.

Glioblastoma (GBM) is the most prevalent and advanced malignant primary brain tumor in adults. GBM frequently harbors epidermal growth factor receptor (EGFR) wild-type (EGFRwt) gene amplification and/or EGFRvIII activating mutation. EGFR-driven GBM relies on the thioredoxin (Trx) and/or glutathione (GSH) antioxidant systems to withstand the excessive production of reactive oxygen species (ROS). The impact of EGFRwt or EGFRvIII overexpression on the response to a Trx/GSH co-targeting strategy is unknown. In this study, we investigated Trx/GSH co-targeting in the context of EGFR overexpression in GBM. Auranofin is a thioredoxin reductase (TrxR) inhibitor, FDA-approved for rheumatoid arthritis. L-buthionine-sulfoximine (L-BSO) inhibits GSH synthesis by targeting the glutamate-cysteine ligase catalytic (GCLC) enzyme subunit. We analyzed the mechanisms of cytotoxicity of auranofin and the interaction between auranofin and L-BSO in U87MG, U87/EGFRwt, and U87/EGFRvIII GBM isogenic GBM cell lines. ROS-dependent effects were assessed using the antioxidant N-acetylsteine. We show that auranofin decreased TrxR1 activity and increased ROS. Auranofin decreased cell vitality and colony formation and increased protein polyubiquitination through ROS-dependent mechanisms, suggesting the role of ROS in auranofin-induced cytotoxicity in the three cell lines. ROS-dependent PARP-1 cleavage was associated with EGFRvIII downregulation in U87/EGFRvIII cells. Remarkably, the auranofin and L-BSO combination induced the significant depletion of intracellular GSH and synergistic cytotoxicity regardless of EGFR overexpression. Nevertheless, molecular mechanisms associated with cytotoxicity were modulated to a different extent among the three cell lines. U87/EGFRvIII exhibited the most prominent ROS increase, P-AKT(Ser-473), and AKT decrease along with drastic EGFRvIII downregulation. U87/EGFRwt and U87/EGFRvIII displayed lower basal intracellular GSH levels and synergistic ROS-dependent DNA damage compared to U87MG cells. Our study provides evidence for ROS-dependent synergistic cytotoxicity of auranofin and L-BSO combination in GBM in vitro. Unraveling the sensitivity of EGFR-overexpressing cells to auranofin alone, and synergistic auranofin and L-BSO combination, supports the rationale to repurpose this promising pro-oxidant treatment strategy in GBM.

A simplified method for preventing postmortem alterations of brain prostanoids for true in situ level quantification

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Standardised and automated assessment of head computed tomography reliably predicts poor functional outcome after cardiac arrest: a prospective multicentre study

PurposeApplication of standardised and automated assessments of head computed tomography (CT) for neuroprognostication after out-of-hospital cardiac arrest.MethodsProspective, international, multicentre, observational study within the Targeted Hypothermia versus Targeted Normothermia after out-of-hospital cardiac arrest (TTM2) trial. Routine CTs from adult unconscious patients obtained > 48 h ≤ 7 days post-arrest were assessed qualitatively and quantitatively by seven international raters blinded to clinical information using a pre-published protocol. Grey–white-matter ratio (GWR) was calculated from four (GWR-4) and eight (GWR-8) regions of interest manually placed at the basal ganglia level. Additionally, GWR was obtained using an automated atlas-based approach. Prognostic accuracies for prediction of poor functional outcome (modified Rankin Scale 4–6) for the qualitative assessment and for the pre-defined GWR cutoff < 1.10 were calculated.Results140 unconscious patients were included; median age was 68 years (interquartile range [IQR] 59–76), 76% were male, and 75% had poor outcome. Standardised qualitative assessment and all GWR models predicted poor outcome with 100% specificity (95% confidence interval [CI] 90–100). Sensitivity in median was 37% for the standardised qualitative assessment, 39% for GWR-8, 30% for GWR-4 and 41% for automated GWR. GWR-8 was superior to GWR-4 regarding prognostic accuracies, intra- and interrater agreement. Overall prognostic accuracy for automated GWR (area under the curve [AUC] 0.84, 95% CI 0.77–0.91) did not significantly differ from manually obtained GWR.ConclusionStandardised qualitative and quantitative assessments of CT are reliable and feasible methods to predict poor functional outcome after cardiac arrest. Automated GWR has the potential to make CT quantification for neuroprognostication accessible to all centres treating cardiac arrest patients.

Growth hormone increase by luteinizing hormone-releasing hormone reflects gonadotroph-related characteristics in acromegaly.

We previously showed the clinical characteristics of acromegaly with a paradoxical growth hormone (GH) response to oral glucose or thyrotropin-releasing hormone. However, the clinical characteristics of acromegaly with an increased GH response to luteinizing hormone-releasing hormone (LHRH responders) remain unclear. The aim of the present study was to evaluate the clinical characteristics, especially gonadotroph-related characteristics of LHRH responders in acromegaly. The clinical characteristics of 33 LHRH responders and 81 LHRH nonresponders were compared. No differences in age, sex or basal serum levels of GH, insulin-like growth factor-1 (IGF-1), and gonadotropin were observed between the two groups. Steroidogenic factor 1 (SF-1), gonadotropin-releasing hormone receptor (GnRHR), and LH expression was more frequently observed in LHRH responders (P < 0.05). In addition, a greater increased rate of GH after LHRH loading, and the proportion of GnRHR and gonadotropin expression was observed in pituitary tumor with SF-1 expression than that without the expression (P < 0.01). LHRH responders showed a greater GH decrease in the octreotide test and a greater IGF-1 decrease after first-generation somatostatin ligand than LHRH nonresponders (P < 0.05). Furthermore, the proportion of hypointense pituitary tumors on T2-weighted magnetic resonance imaging and tumors with densely granulated type was higher in LHRH responders than in LHRH nonresponders, respectively (P < 0.05). No difference between the two groups was observed in either somatostatin receptor 2 or 5 expression. The increased GH response to LHRH is associated with the gonadotroph-related characteristics. This response may reflect the biological characteristics of somatotroph tumors.

Altered Hippocampal Glutamatergic Neurotransmission and Cognitive Impairment in APP Knock-In Mice.

It is well established that glutamatergic neurotransmission plays an essential role in learning and memory. Previous studies indicate that glutamate dynamics shift with Alzheimers disease (AD) progression, contributing to negative cognitive outcomes. In this study, we characterized hippocampal glutamatergic signaling with age and disease progression in a knock-in mouse model of AD (APPNL-F/NL-F). At 2-4 and 18+ months old, male and female APPNL/NL, APPNL-F/NL-F, and C57BL/6 mice underwent cognitive assessment using Morris water maze (MWM) and Novel Object Recognition (NOR). Then, basal and 70 mM KCl stimulus-evoked glutamate release was measured in the dentate gyrus (DG), CA3, and CA1 regions of the hippocampus using a glutamate-selective microelectrode in anesthetized mice. Glutamate recordings support elevated stimulus-evoked glutamate release in the DG and CA3 of young APPNL-F/NL-F male mice that declined with age compared to age-matched control mice. Young female APPNL-F/NL-F mice exhibited increased glutamate clearance in the CA1 that slowed with age compared to age-matched control mice. Male and female APPNL-F/NL-F mice exhibited decreased CA1 basal glutamate levels, while males also showed depletion in the CA3. Cognitive assessment demonstrated impaired spatial cognition in aged male and female APPNL-F/NL-F mice, but only aged females displayed recognition memory deficits compared to age-matched control mice. These findings confirm a sex-dependent hyper-to-hypoactivation glutamatergic paradigm in APPNL-F/NL-F mice. Further, data illustrate a sexually dimorphic biological aging process resulting in a more severe cognitive phenotype for female APPNL-F/NL-F mice than their male counterparts. Research outcomes mirror that of human AD pathology and provide further evidence of divergent AD pathogenesis between sexes.

Long-term changes in the phenotype and cytokine production of monocytes in COVID-19 recovered and vaccinated individuals.

Monocytes play a crucial role in the immune response against pathogens. Here, we sought to determine COVID-19 and the vaccine Gam-COVID-Vac induce long-term changes in the phenotype and cytokine production of circulating monocytes. Monocytes were purified from peripheral blood mononuclear cells of healthy donors who had not had COVID-19 or vaccination, who had received two doses of Gam-COVID-Vac, and who had mild/moderate COVID-19 in the last 6 months and evaluated by flow cytometry. To investigate the effect of SARS-CoV-2 proteins, monocytes were cultured for 2 days with or without stimulation with recombinant SARS-CoV-2 S1 and N peptides. Monocytes obtained from vaccinated and recovered individuals showed increased basal expression of HLA-DR, CD63, CXCR2, and TLR7. We also observed an increased frequency of CD63+ classical monocytes in both groups, as well as an increased frequency of HLA-DR+ non-classical monocytes in the COVID-19-recovered group compared to the control group. Monocytes from vaccinated and recovered donors produced higher basal levels of IL-6, IL-1β, and TNF-α cytokines. Ex vivo stimulation with SARS-CoV-2 antigens induced increased expression of HLA-DR and TLR7 on monocytes obtained from the control group. The challenge with SARS-CoV-2 antigens had no effect on the production of IL-6, IL-1β, and TNF-α cytokines by monocytes. The acquired data offer compelling evidence of enduring alterations in both the phenotype and functional status of circulating monocytes subsequent to vaccination with Gam-COVID-Vac and mild/moderate COVID-19 infection. At least some of these changes appear to be a consequence of exposure to SARS-CoV-2 S1 and N antigens.

Ultrasound Therapy With High-Pressure Pulses Is Effective to Reduce the Effects of Collagenase-Induced Tendinopathy in Rat's Achilles Tendon

ObjectiveWe propose an ultrasonic treatment for collagenase-induced tendinopathy in rat's Achilles tendon using pulses with a low number of cycles, high acoustic pressure and very low duty cycle. MethodsTwenty rats were used to perform the experiment. Four experimental groups of calcaneal tendons were studied: control (n = 6), sham (n = 4), collagenase-induced tendinopathy (n = 8) and ultrasound-treated collagenase-induced tendinopathy (n = 8). Surgical intervention was performed to expose the tendons prior to collagenase injection. A 1 MHz ultrasonic tansducer with a focusing lens was used. Ultrasonic treatments were used with an average total treatment time of 2.5 min, 20-cycle pulses, pressure amplitude p = 7 MPa, and 0.02% duty cycle. Histopathology of the samples was performed to evaluate nuclear density, acute inflammation, and signs of neovascularization. Collagen (types I and III), elastic fibers, and glycosaminoglycans were also analyzed. ResultsNo tendon involvement was found by the surgical process. Ultrasonic treatment is safe, as it does not affect healthy tendons. When collagenase infiltrated animals were treated with US, a clear predominance of type I collagen fibers and a similar collagen ratio profile to that observed in the control and sham groups was observed, with a higher density of elastic fibers compared to the control and sham groups and a significant increase in the density of glycosaminoglycans. ConclusionThe ultrasound treatment proposed reduces the effects of the artificial collagenase lesion to reach the basal level after 45 d.