Factor Activity Research Articles

Early Online.

Long-haul flights have been associated with a two- to four-fold increased risk of aviation-related thrombosis (ART). Several studies have investigated the extent to which hypoxic hypobaric exposure, dehydration and prolonged immobilisation during air travel induce changes in haemostasis. To investigate the role of high altitude as a risk factor for ART. Healthy volunteers aged ≥18 years (N=40), without risk factors for venous thromboembolism, were exposed to an exacerbated altitude of 18 000 feet (5 486 m) for 1 hour. During the flight, the oxygen (O2) levels of the participants, who received supplemental O2, were measured by pulse oximetry and maintained at >92%. Venous blood and urine samples were collected prior to departure and immediately after flying in an unpressurised twin-engine airplane. D-dimer levels, thromboelastography (TEG) parameters, von Willebrand factor (VWF) activity and urine osmolality were measured. The participants were 19 men and 21 women, with a mean (standard deviation) age of 46 (14) years. A significant difference in D-dimer levels, VWF activity, urine osmolality and TEG parameters (reaction (R) time, kinetic (K) time and maximum amplitude (MA)) before and after the 1-hour flight was observed (p<0.001). Urine osmolality correlated positively with VWF activity levels (r=0.469; p<0.002). Air travel at high altitude induced a hypercoagulable state in healthy volunteers. Future research should focus on whether thromboprophylaxis can significantly obviate the activation of coagulation in response to high altitude.

YTHDF2 Regulates Advanced Glycation End Products-Induced Melanogenesis through Inhibiting A20 Expression in Human Dermal Fibroblasts.

Fibroblast A20 suppresses advanced glycation end products (AGEs)-induced melanogenesis by inhibiting NLRP3 inflammasome activation. AGEs repress A20 expression and significantly m6A-methylate A20 mRNA in fibroblasts. YTHDF2 is the most studied m6A reader protein and can accelerate degradation of m6A-modified mRNA. Whether YTHDF2 regulates AGEs-induced A20 expression and pigmentation is unknown. In this study, we confirmed that YTHDF2 inversely regulated AGEs-BSA-inhibited A20 expression but facilitated AGEs-BSA-activated NF-κB signaling and NLRP3 inflammasome in fibroblasts via YTHDF2 knockdown and overexpression experiments. Mechanistically, YTHDF2 bound to m6A-modified A20 mRNA induced by AGEs-BSA and increased its degradation. Moreover, fibroblast YTHDF2 robustly promoted AGEs-BSA-induced IL-18 level in coculture supernatants and melanin content, tyrosinase activity, and expression of microphthalmia-associated transcription factor and tyrosinase in melanocytes, which were significantly blocked by IL-18 binding protein. Further, fibroblast YTHDF2 markedly increased AGEs-BSA-induced epidermal melanin level in cocultured ex vivo skin and MAPKs activation in melanocytes. Importantly, upregulated dermal YTHDF2 expression was negatively correlated with dermal A20 level and positively associated with both epidermal melanin and dermal AGEs content in sun-exposed skin and lesions of melasma and solar lentigo. These findings suggest that fibroblast YTHDF2 positively regulates AGEs-induced melanogenesis mainly via A20/ NF-κB /NLRP3 inflammasome/ IL-18 /MAPKs axis in an m6A-dependent manner and functions in photoaging-induced hyperpigmentation skin disorders.

Low-oxygen-induced root bending is altered by phytoglobin1 through mediation of ethylene response factors (ERFs) and auxin signaling.

phytoglobin1 positively regulates root bending in hypoxic Arabidopsis roots through regulation of ethylene response factors and auxin transport. Hypoxia-induced root bending is known to be mediated by the redundant activity of the group VII ethylene response factors (ERFVII) RAP2.12 and HRE2, causing changes in polar auxin transport (PAT). Here, we show that phytoglobin1 (Pgb1), implicated in hypoxic adaptation through scavenging of nitric oxide (NO), can alter root direction under low oxygen. Hypoxia-induced bending is exaggerated in roots over-expressing Pgb1 and attenuated in those where the gene is suppressed. These effects were attributed to Pgb1 repressing both RAP2.12 and HRE2. Expression, immunological and genetic data place Pgb1 upstream of RAP2.12 and HRE2 in the regulation of root bending in oxygen-limiting environments. The attenuation of slanting in Pgb1-suppressing roots was associated with depletion of auxin activity at the root tip because of depression in PAT, while exaggeration of root bending in Pgb1-over-expressing roots with the retention of auxin activity. Changes in PIN2 distribution patterns, suggestive of redirection of auxin movement during hypoxia, might contribute to the differential root bending responses of the transgenic lines. In the end, Pgb1, by regulating NO levels, controls the expression of 2 ERFVIIs which, in a cascade, modulate PAT and, therefore, root bending.

Prediction model for leaflet thrombosis in patients undergoing transcatheter aortic valve implantation: the EFFORT study.

Leaflet thrombosis (LT) isa multifaceted and underexplored condition that can manifest following transcatheter aortic valve implantation (TAVI). The objective of this study was to formulate a prediction model based on laboratory assessments and clinical parameters, providing additional guidance and insight into this relatively unexplored aspect of post-TAVI complications. The present study was an observational prospective hypothesis-generating study, including 101 patients who underwent TAVI and a screening for LT (the primary endpoint) by multidetector computed tomography (MDCT). All images were acquired on a third-generation dual-source CT system. Levels of von Willebrand factor (vWF) activity, hemoglobin (Hb), and lactate dehydrogenase (LDH) were measured among other parameters. A predictive score utilizing binary logistic regression, Kaplan-Meier time-to-event analysis, and receiver operating characteristics (ROC) analysis was established. LT (11 subclinical and 2 clinical) was detected in 13 of 101 patients (13%) after a median time to screening by MDCT of 105days (IQR, 98-129days). Elevated levels of vWF activity (> 188%) pre-TAVI, decreased Hb values (< 11.9g/dL), as well as increased levels of LDH (> 312 U/L) post-TAVI and absence of oral anticoagulation (OAC) were found in patients with subsequent LT formation as compared to patients without LT. The established EFFORT score ranged from - 1 to 3 points, with an increased probability for LT development in patients with ≥ 2 points (85.7% of LT cases) vs < 2 points (14.3% of LT cases; p < 0.001). Achieving an EFFORT score of ≥ 2 points was found to be significantly associated with a 10.8 times higher likelihood of developing an LT (p = 0.001). The EFFORT score has an excellent c-statistic (area under the curve (AUC) = 0.89; 95% CI 0.74-1.00; p = 0.001) and a high negative predictive value (98%). An EFFORT score might be a helpful tool to predict LT development and could be used in risk assessment, if validated in confirmatory studies. Therefore, the score has the potential to guide the stratification of individuals for the planning of subsequent MDCT screenings.

Molecular subtypes explain lupus epigenomic heterogeneity unveiling new regulatory genetic risk variants

The heterogeneity of systemic lupus erythematosus (SLE) can be explained by epigenetic alterations that disrupt transcriptional programs mediating environmental and genetic risk. This study evaluated the epigenetic contribution to SLE heterogeneity considering molecular and serological subtypes, genetics and transcriptional status, followed by drug target discovery. We performed a stratified epigenome-wide association studies of whole blood DNA methylation from 213 SLE patients and 221 controls. Methylation quantitative trait loci analyses, cytokine and transcription factor activity - epigenetic associations and methylation-expression correlations were conducted. New drug targets were searched for based on differentially methylated genes. In a stratified approach, a total of 974 differential methylation CpG sites with dependency on molecular subtypes and autoantibody profiles were found. Mediation analyses suggested that SLE-associated SNPs in the HLA region exert their risk through DNA methylation changes. Novel genetic variants regulating DNAm in disease or in specific molecular contexts were identified. The epigenetic landscapes showed strong association with transcription factor activity and cytokine levels, conditioned by the molecular context. Epigenetic signals were enriched in known and novel drug targets for SLE. This study reveals possible genetic drivers and consequences of epigenetic variability on SLE heterogeneity and disentangles the DNAm mediation role on SLE genetic risk and novel disease-specific meQTLs. Finally, novel targets for drug development were discovered.

HOP stabilizes the HSFA1a and plays a main role in the onset of thermomorphogenesis.

Living organisms have the capacity to respond to environmental stimuli, including warm conditions. Upon sensing mild temperature, plants launch a transcriptional response that promotes morphological changes, globally known as thermomorphogenesis. This response is orchestrated by different hormonal networks and by the activity of different transcription factors, including the heat shock factor A1(HSFA1) family. Members of this family interact with heat shock protein70(HSP70) and heat shock protein 90(HSP90); however, the effect of this binding on the regulation of HSFA1 activity or of the role ofcochaperones, such as the HSP70-HSP90 organizing protein (HOP) on HSFA1 regulation, remains unknown.Here, we show that AtHOPs are involved in the folding and stabilization of the HSFA1a and are required for the onset of the transcriptional response associated to thermomorphogenesis. Our results demonstrate that the three members of the AtHOP family bind in vivo to the HSFA1a and that the expression of multiple HSFA1a-responsive-responsive genes is altered in the hop1 hop2 hop3 mutant under warm temperature. Interestingly, HSFA1a is accumulated at lower levels in the hop1 hop2 hop3 mutant, while control levels are recovered in the presence of the proteasome inhibitor MG132 or the synthetic chaperone tauroursodeoxycholic acid (TUDCA). This uncovers the HSFA1a as a client of HOP complexes in plants and reveals the participation of HOPs in HSFA1a stability.

YAP/TAZ interacts with RBM39 to confer resistance against indisulam

The Hippo pathway and its downstream effectors, Yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ), are essential for cell growth and organ development. Emerging evidence revealed that the Hippo pathway and YAP/TAZ are frequently dysregulated by multiple genetic alterations in solid cancers including head and neck squamous cell carcinoma (HNSCC); however, the YAP/TAZ-nuclear interactome remains unclear. RNA-binding motif protein 39 (RBM39) enhances transcriptional activity of several transcription factors and also regulates mRNA splicing. Indisulam degrading RBM39 induces alternative splicing, leading to cell death. However, clinical trials of indisulam have failed to show effectiveness. Therefore, clarifying the resistance mechanism against splicing inhibitors is urgently required. In this study, we identified RBM39 as a novel YAP/TAZ-interacting molecule by proteome analysis. RBM39 promoted YAP/TAZ transcriptional activity. We further elucidated that indisulam reduces RBM39/YAP/TAZ-mediated integrin or collagen expression, thereby inactivating focal adhesion kinase (FAK) important for cell survival. Moreover, indisulam also induced alternative splicing of cell cycle- or DNA metabolism-related genes. YAP/TAZ hyperactivation delayed indisulam-induced RBM39 degradation, which restored the integrin/collagen expression to activate FAK, and alternative splicing, thereby conferring resistance against indisulam in vitro and in vivo. Our findings may aid to develop a novel cancer therapy focusing on YAP/TAZ/RBM39 interaction.

Studies on the potential risk of amyloidosis from exposure to cultured fibril from silk fibroin

Amyloid A (AA) amyloidosis is induced by administering amyloid fibrils to animals under inflammatory conditions. Silk fibroin (SF), the main component of silk threads, forms amyloid-like fibrils and has been previously reported to induce AA amyloidosis in mice. In this study, SF was cultured in ethanol solution, and after confirming fibril formation through thioflavin T assay, Congo red assay, and observation under electron microscopy, cultured SF ethanol solutions were administered to mice via various routes to investigate the induction of target organs and amyloidosis. As a result, cultured SF ethanol solutions were confirmed to reach the lungs and spleen, but no amyloid deposition was observed. While SF forms amyloid-like fibril structures through cultivation in ethanol solution, its amyloid-enhancing factor (AEF) activity is considered low in mice.

SoxB1 transcription factors are essential for initiating and maintaining neural plate border gene expression.

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

An integrated approach to identifying sex-specific genes, transcription factors, and pathways relevant to Alzheimer's disease

BackgroundAge represents a significant risk factor for the development of Alzheimer's disease (AD); however, recent research has documented an influencing role of sex in several features of AD. Understanding the impact of sex on specific molecular mechanisms associated with AD remains a critical challenge to creating tailored therapeutic interventions. MethodsThe exploration of the sex-based differential impact on disease (SDID) in AD used a systematic review to first select transcriptomic studies of AD with data regarding sex in the period covering 2002 to 2021 with a focus on the primary brain regions affected by AD - the cortex (CT) and the hippocampus (HP). A differential expression analysis for each study and two tissue-specific meta-analyses were then performed. Focusing on the CT due to the presence of significant SDID-related alterations, a comprehensive functional characterization was conducted: protein-protein network interaction and over-representation analyses to explore biological processes and pathways and a VIPER analysis to estimate transcription factor activity. ResultsWe selected 8 CT and 5 HP studies from the Gene Expression Omnibus (GEO) repository for tissue-specific meta-analyses. We detected 389 significantly altered genes in the SDID comparison in the CT. Generally, female AD patients displayed more affected genes than males; we grouped said genes into six subsets according to their expression profile in female and male AD patients. Only subset I (repressed genes in female AD patients) displayed significant results during functional profiling. Female AD patients demonstrated more significant impairments in biological processes related to the regulation and organization of synapsis and pathways linked to neurotransmitters (glutamate and GABA) and protein folding, Aβ aggregation, and accumulation compared to male AD patients. These findings could partly explain why we observe more pronounced cognitive decline in female AD patients. Finally, we detected 23 transcription factors with different activation patterns according to sex, with some associated with AD for the first time. All results generated during this study are readily available through an open web resource Metafun-AD (https://bioinfo.cipf.es/metafun-ad/). ConclusionOur meta-analyses indicate the existence of differences in AD-related mechanisms in female and male patients. These sex-based differences will represent the basis for new hypotheses and could significantly impact precision medicine and improve diagnosis and clinical outcomes in AD patients.

SSR42 is a Novel Regulator of Cytolytic Activity in Staphylococcus aureus.

S. aureus is a master pathogen due to its formidable collection of virulence factors. These are tightly controlled by a diverse group of regulators that titrate their abundance to adapt to unique infectious niches. The role of regulatory RNAs in stress adaptation and pathogenesis is becoming increasingly more relevant in S. aureus . In this study, we provide the most comprehensive global analysis to date of just such a factor, SSR42. Specifically, we uncover that SSR42 is required for mediating cytotoxicity - one of the pillars of infection - in response to phagocytosis by human neutrophils. We find that SSR42 is induced by components of the host immune system and facilitates downstream activation of cytotoxic factors via RNA-RNA interactions. This illustrates that SSR42 forms a pivotal link between sensing the external environment and mediating resistance to oxidative stress while promoting virulence, solidifying it as a major global regulator in S. aureus .

Sensing and guiding cell-state transitions by using genetically encoded endoribonuclease-mediated microRNA sensors.

Precisely sensing and guiding cell-state transitions via the conditional genetic activation of appropriate differentiation factors is challenging. Here we show that desired cell-state transitions can be guided via genetically encoded sensors, whereby endogenous cell-state-specific miRNAs regulate the translation of a constitutively transcribed endoribonuclease, which, in turn, controls the translation of a gene of interest. We used this approach to monitor several cell-state transitions, to enrich specific cell types and to automatically guide the multistep differentiation of human induced pluripotent stem cells towards a haematopoietic lineage via endothelial cells as an intermediate state. Such conditional activation of gene expression is durable and resistant to epigenetic silencing and could facilitate the monitoring of cell-state transitions in physiological and pathological conditions and eventually the 'rewiring' of cell-state transitions for applications in organoid-based disease modelling, cellular therapies and regenerative medicine.

Predictive and therapeutic value of lipoprotein-associated phospholipaseA2 in sarcopenia in chronic obstructive pulmonary disease

BackgroundSarcopenia, characterized by progressive muscle dysfunction, is a common complication of chronic obstructive pulmonary disease (COPD). Our previous study revealed serum Lipoprotein-associated phospholipaseA2 (Lp-PLA2) level significantly increased in COPD and associated with exercise tolerance. This study further investigated the functions and target potential of Lp-PLA2 for sarcopenia in COPD. MethodsThe circulating Lp-PLA2 level/enzyme activity in COPD patients and age-matched healthy volunteers were measured. Clinical parameters on skeletal muscle were measured and their correlations with Lp-PLA2 were analyzed. We explored the involvement of Lp-PLA2 in vivo and treatment effectiveness of darapladib (a specific Lp-PLA2 inhibitor) in CS-induced muscle dysfunction models. ResultsCirculating Lp-PLA2 level/enzyme activity was elevated in COPD patients compared with healthy controls, negatively associated with skeletal muscle mass and function. In CS-induced muscle dysfunction murine models, up-regulated serum Lp-PLA2 level/enzyme activity was verified again. In CS-exposed mouse models, darapladib treatment reversed muscle mass loss and muscle dysfunction, meanwhile rescued upregulation of MuRF1 and atrogin-1, and activation of inflammatory factors, oxidant enzymes and NF-κB signaling. ConclusionsLp-PLA2 could be a potential indicator for sarcopenia in COPD. Darapladib, a Lp-PLA2 inhibitor, can alleviate CS-induced skeletal muscle dysfunction and represents a potential therapeutic for sarcopenia in COPD.

Protein Phosphatase 2ACα Regulates ATR-Mediated Endogenous DNA Damage Response Against Microcephaly.

Protein phosphatase 2A (PP2A) is an abundant heterotrimeric holoenzyme in eukaryotic cells coordinating with specific kinases to regulate spatial-temporal protein dephosphorylation in various biological processes. However, the function of PP2A in cortical neurogenesis remains largely unknown. Here, we report that neuronal-specific deletion of Pp2acα in mice displayed microcephaly, with significantly smaller brains and defective learning and memory ability. Mechanistically, neuronal Pp2acα deficiency resulted in elevated endogenous DNA damage and activation of ATR/CHK1 signaling. It was further induced by the loss of direct interaction between PP2AC and ATR as well as the function of PP2AC to dephosphorylate ATR. Importantly, ATR/CHK1 signaling dysregulation altered both the expression and activity of several critical downstream factors including P53, P21, Bcl2, and Bax, which led to decreased proliferation of cortical progenitor cells and increased apoptosis in developing cortical neurons. Taken together, our results indicate an essential function of PP2ACα in endogenous DNA damage response-mediated ATR signaling during neurogenesis, and defective PP2ACα in neurons contributes to microcephaly.

Molecular analysis of a self-organizing signaling pathway for Xenopus axial patterning from egg to tailbud

Xenopus embryos provide a favorable material to dissect the sequential steps that lead to dorsal-ventral (D-V) and anterior-posterior (A-P) cell differentiation. Here, we analyze the signaling pathways involved in this process using loss-of-function and gain-of-function approaches. The initial step was provided by Hwa, a transmembrane protein that robustly activates early β-catenin signaling when microinjected into the ventral side of the embryo leading to complete twinned axes. The following step was the activation of Xenopus Nodal-related growth factors, which could rescue the depletion of β-catenin and were themselves blocked by the extracellular Nodal antagonists Cerberus-Short and Lefty. During gastrulation, the Spemann-Mangold organizer secretes a cocktail of growth factor antagonists, of which the BMP antagonists Chordin and Noggin could rescue simultaneously D-V and A-P tissues in β-catenin-depleted embryos. Surprisingly, this rescue occurred in the absence of any β-catenin transcriptional activity as measured by β-catenin activated Luciferase reporters. The Wnt antagonist Dickkopf (Dkk1) strongly synergized with the early Hwa signal by inhibiting late Wnt signals. Depletion of Sizzled (Szl), an antagonist of the Tolloid chordinase, was epistatic over the Hwa and Dkk1 synergy. BMP4 mRNA injection blocked Hwa-induced ectopic axes, and Dkk1 inhibited BMP signaling late, but not early, during gastrulation. Several unexpected findings were made, e.g., well-patterned complete embryonic axes are induced by Chordin or Nodal in β-catenin knockdown embryos, dorsalization by Lithium chloride (LiCl) is mediated by Nodals, Dkk1 exerts its anteriorizing and dorsalizing effects by regulating late BMP signaling, and the Dkk1 phenotype requires Szl.

An activity-specificity trade-off encoded in human transcription factors

Transcription factors (TFs) control specificity and activity of gene transcription, but whether a relationship between these two features exists is unclear. Here we provide evidence for an evolutionary trade-off between the activity and specificity in human TFs encoded as submaximal dispersion of aromatic residues in their intrinsically disordered protein regions. We identified approximately 500 human TFs that encode short periodic blocks of aromatic residues in their intrinsically disordered regions, resembling imperfect prion-like sequences. Mutation of periodic aromatic residues reduced transcriptional activity, whereas increasing the aromatic dispersion of multiple human TFs enhanced transcriptional activity and reprogramming efficiency, promoted liquid–liquid phase separation in vitro and more promiscuous DNA binding in cells. Together with recent work on enhancer elements, these results suggest an important evolutionary role of suboptimal features in transcriptional control. We propose that rational engineering of amino acid features that alter phase separation may be a strategy to optimize TF-dependent processes, including cellular reprogramming.

A single-cell transcriptomic study of heterogeneity in human embryonic tanycytes

Disruptions in energy homeostasis can lead to diseases like obesity and diabetes, affecting millions of people each year. Tanycytes, the adult stem cells in the hypothalamus, play crucial roles in assisting hypothalamic neurons in maintaining energy balance. Although tanycytes have been extensively studied in rodents, our understanding of human tanycytes remains limited. In this study, we utilized single-cell transcriptomics data to explore the heterogeneity of human embryonic tanycytes, investigate their gene regulatory networks, analyze their intercellular communication, and examine their developmental trajectory. Our analysis revealed the presence of two clusters of β tanycytes and three clusters of α tanycytes in our dataset. Surprisingly, human embryonic tanycytes displayed significant similarities to mouse tanycytes in terms of marker gene expression and transcription factor activities. Trajectory analysis indicated that α tanycytes were the first to be generated, giving rise to β tanycytes in a dorsal–ventral direction along the third ventricle. Furthermore, our CellChat analyses demonstrated that tanycytes generated earlier along the developmental lineages exhibited increased intercellular communication compared to those generated later. In summary, we have thoroughly characterized the heterogeneity of human embryonic tanycytes from various angles. We are confident that our findings will serve as a foundation for future research on human tanycytes.

P-104 Cytoprotective effect of melatonin in prepubertal Sertoli cells: role in cadmium-induced idiopathic male infertility

Abstract Study question Melatonin (MLT) is a cytoprotective agent holding potential to prevent cadmium (Cd) toxicity and its impact in testicular function and fertility. Summary answer MLT produced a potent cytoprotective effect in Sertoli cells (SCs) exposed to Cd-toxicity, reducing its H2O2 generation and reductive stress effects by modulation Nrf2 activity. What is known already MLT a hormonal substance exhibiting strong antioxidative and antiapoptotic properties, has been proved to hold great potential as cytoprotec tive agent in a range of tissues and cell models exposed to Cd toxicity, including the rat bone, ovaries, human MSC, and even mammalian testes. However, cellular and mechanistic aspects of this effect of MLT in testes remain poorly understood. Cd is a pro-oxidant heavy metal reported to interfere with testicular function and fertility. Both the toxicity of Cd and the cytoprotective function of MLT involve redox-dependent mechanisms with the activity of Nrf2 transcription factor as key player on the cellular stress response. Study design, size, duration Porcine pre-pubertal SCs were maintained at 37 °C in a 5% CO2 humidified atmosphere in the absence (unexposed-control group) or presence of 5 or 10µM CdCl2 (Sigma Chemical Co.) and/or 50nM MLT (Sigma-Aldrich) for 48h in HAMF12 (Euroclone) supplemented with 0.166nM retinoic acid (Sigma-Aldrich Co.) and 5/500ml of Insulin-Transferrin-Selenium (ITS) + Premix (Cat. No. 354352; Corning). Participants/materials, setting, methods After treatment the parameters investigated in the study were: a) SCs viability (MTT test); b) SCs functionality parameters AMH and inhibin B. (real-time PCR and ELISA analysis); c) Intracellular and extracellular ROS production ( with Amplex Red and DCFH-DA fluorescent probe respectively); d) Catalase enzyme activity (spectrophotometric assay procedure); e) Nrf2 and other transcription factors (by semi-quantitative microplate confocal microscopy analysis); f) Intracellular and extracellular levels of GSH and other thiols (by HPLC); g) c-Jun and ERK1/2 (Immunoblot). Main results and the role of chance MLT co-treatment significantly prevented the effects of Cd on cell viability and abnormal ROS production. The reduction of H2O2 efflux was very important and combined with an increased CAT activity that was demonstrated upon MLT treatment significantly in SCs exposed to 5μMCd. The co-treatment with MLT compared to Cd treatment, showed a trend toward a significant recovery of AMH and inhibin B gene expression and secretion levels, respectively, demonstrating the cytoprotective effect of MLT in SCs exposed to Cd toxicity at the functional level. In Cd-treated cells, MLT significantly reduced the expression and nuclear translocation of Nrf2 protein, and decreased GSTP induction. However, MLT reduced Cys uptake and stimulated GSH efflux when the cells were treated with 10 µM Cd, suggesting a specific role for the Nrf2-GSTP axis in the cytoprotective response to MLT of Cd-treated SCs. Finally MLT induced MAPK-ERK1-2 pathway and c-Jun transcription factor when the cells were treated with Cd. MLT is a cytoprotective agent in SCs exposed to Cd toxicity capable to restore cell viability and function. Its activity is associated with the modulation of Nrf2 and other stress response genes important in the control of thiols and H2O2 metabolism of SCs, including cJun and MAPKs. Limitations, reasons for caution Other ROS-scavenging genes other than CAT may have a role in the antioxidant response to MLT of SCs, such as superoxide dismutase, glutathione peroxidases, and peroxyredoxins that are worth investigating further. Wider implications of the findings The aspects that emerged from our study and, described for the first time in these cells, warrant further preclinical and clinical investigation holding potential in the treatment of Cd-induced testicular damage and fertility problems. Trial registration number Not applicable

Knockdown of LincRNACOX2 Alleviates Oxidative Stress in Pathophysiology of Acute Lung Injury

Acute lung injury (ALI) has a complicated etiology that involves oxidative stress and inflammation. The role of lncRNACox2 (lincCOX2) in ALI regulation remains unclear. In this study, the ALI model of mice and MLE-12 cell was induced by LPS. To investigate the expression of lncRNACox2 in these ALI models, we employed a nanomagnetic bead-based RNA extraction method for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. This enabled us to determine the levels of lncRNACox2 expression and evaluate knockdown efficiency. Moreover, we also examined lung tissue histopathology using H&amp;E staining. Cell survival and apoptosis rates were evaluated through CCK-8 and flow cytometry, respectively. The concentration of inflammatory factors was measured using ELISA. Additionally, the concentration (8-OHdG and MDA) and enzymatic activity (CAT, GSH-Px, and SOD) of oxidative stress related factors were measured by biochemical method. The western blot was performed to present the key proteins expression level in Nrf2/ARE signaling pathway in cytoplasm and nucleoprotein. The ALI mouse model was successfully established. The histopathology change and inflammatory cells were observed by H&amp;E staining in LPS treated groups. The expression of lincCOX2 was up-regulated in ALI tissue. LPS induced more cell death in ALI, and the knockdown of lincCOX2 improved the cell survival and suppressed the apoptosis in ALI cell. Furthermore, In addition, downregulation of lincCOX2 attenuated inflammation and oxidative stress in lung cells in ALI. The concentration of 8-OHdG and MDA were highest in the LPS group while reduced by the sh-lincCOX2, the activity of CAT, GSH-Px, and SOD was reduced in the LPS induced ALI and increased by the sh-lincCOX2. In ALI, the distribution of Nrf2 protein is transferred from cytoplasm to nucleus. Furthermore, the lincCOX2 regulated oxidative stress via Nrf2/ARE signaling pathway in ALI. Overall, downregulation of lincRNACOX2 alleviates oxidative stress in ALI via Nrf2/ARE Pathway. This study suggests that lincCOX2 may be a potential target for the treatment of ALI.

Investigating the Underlying Molecular Mechanisms of Yunke on Bone Metastases from Prostate Cancer.

To explore analgesic effect and bone repair mechanism of non-radioactive technetium-99 conjugated with methylene diphosphonate (99Tc-MDP, brand name, Yunke) on bone metastases (BM). In vivo experiment, mouse BM models of prostate cancer RM-1 cell were constructed and divided into Control, Yunke, 99Tc+SnCl2 and MDP groups based on medicine composition. Tumor specimens were inspected for size, X-ray, microCT and histopathology. In vitro experiment, with Cell Counting Kit-8 (CCK8), scratch, clone, apoptosis, Polymerase Chain Reaction (PCR) and Western Blot experiments, effects of Yunke on RM-1 cells and osteoclast-related cells were observed. In vivo experiment, there was no difference in tumor size between Yunke and control group. Contrasted with control group, in Yunke group, trabecular spacing (Tb.Sp) of tumor bone was lower, bone volume/total volume (BV/TV) on marrow cavity and bone cortex were higher. Tunnel staining showed that positive rate of apoptosis in Yunke group was higher than that in control group. Ki67 staining showed that Yunke could not inhibit proliferation of tumor cells. In vitro experiment, CCK8 and scratch experiments showed that Yunke neither can inhibit proliferation nor can inhibit migration of RM-1 cells. High concentration of Yunke promoted late apoptosis of RM-1 cells. Yunke could inhibit BMM cell proliferation, differentiation of osteoclasts, and osteoclast-related transcription factors. Yunke displayed different degrees of inhibitory effects on MAPKs signaling pathway during osteoclast differentiation. It had obvious inhibitory effects on osteoclast-related transcription factors, such as cFOS, NFATC1, ACP-5, CTSK, D2 and MMP-9, the strongest inhibitory effects were observed with ACP-5, CTSK and D2. Yunke also displayed different degrees of inhibitory effects on protein activities of JNK, pERK, ERK and pP38. Yunke cannot inhibit the proliferation and migration of RM-1 cells, so we think it is not recommended for the treatment of primary tumors and prevention of occurrence of tumors metastatic to bones. The mechanism of therapeutic effect of Yunke on BM by inhibiting proliferation of BMM, inhibiting MAPKs signal transduction and activation of transcription factors during differentiation process of BMM-derived osteoclasts, inhibiting number and size of osteoclasts, inhibiting bone resorption and protecting bone destruction through enhancing bone hardness and bone mass. Thereby, we believe that Yunke is more suitable for promoting the repair induced by BMs, delaying its progression and reducing the occurrence of SREs.