The transcription factors NR5A1 and JUNB cooperate to activate the Axl promoter in mouse Sertoli cell lines.

BACKGROUND: The Axl gene is a receptor tyrosine kinase essential for male fertility. With other Tyro3 family members, it regulates cell apoptosis and preserves the organization of seminiferous tubules. However, the regulation of the expression of Axl in testicular Sertoli cells is not entirely understood. The transcription factors NR5A1 and JUNB are involved in several male fertility mechanisms such as sex development and steroidogenesis. We hypothesize that Axl promoter activity is regulated by cooperation between JUNB and NR5A1 in Sertoli cells. METHODS AND RESULTS: Following transfections of TM4 Sertoli cells with DsiRNA interference against Axl, our results show that cell morphology may be regulated by AXL. Using transfections of expression plasmids and reporter plasmids containing the Axl promoter, we report that Axl expression is highly activated by cooperation between NR5A1 and JUNB in TM4 and 15P-1 Sertoli cells. Chromatin immunoprecipitation and luciferase reporter assays with 5' promoter deletions demonstrate that JUNB and NR5A1 are being recruited to DNA regulatory elements in the proximal region of the Axl promoter. The fourth intronic region of Axl also participates in the recruitment of JUNB. CONCLUSION: Thus, Axl expression is regulated by a cooperation between the transcription factors JUNB and NR5A1 and influences the morphology of TM4 Sertoli cells.

Effects of Red Sorghum-Derived Deoxyanthocyanidins and Their O-ß-D-Glucosides on E-Cadherin Promoter Activity in PC-3 Prostate Cancer Cells.

Although much less common than anthocyanins, 3-Deoxyanthocyanidins (3-DAs) and their glucosides can be found in cereals such as red sorghum. It is speculated that their bioavailability is higher than that of anthocyanins. Thus far, little is known regarding the therapeutic effects of 3-DAs and their O-ß-D-glucosides on cancer, including prostate cancer. Thus, we evaluated their potential to decrease cell viability, to modulate the activity of transcription factors such as NFκB, CREB, and SOX, and to regulate the expression of the gene CDH1, encoding E-Cadherin. We found that 4',7-dihydroxyflavylium chloride (P7) and the natural apigeninidin can reduce cell viability, whereas 4',7-dihydroxyflavylium chloride (P7) and 4'-hydroxy-7-O-ß-D-glucopyranosyloxyflavylium chloride (P3) increase the activities of NFkB, CREB, and SOX transcription factors, leading to the upregulation of CDH1 promoter activity in PC-3 prostate cancer cells. Thus, these compounds may contribute to the inhibition of the epithelial-to-mesenchymal transition in cancer cells and prevent the metastatic activity of more aggressive forms of androgen-resistant prostate cancer.

Regulation of Cdh2 by the AP-1 family transcription factor Junb in TM4 Sertoli cells.

Cadherins are transmembrane proteins that mediate cell-to-cell adhesion and various cellular processes. In Sertoli cells of the testis, Cdh2 contributes to the development of the testis and the formation of the blood-testis barrier, being essential for germ cells' protection. Analyses of chromatin accessibility and epigenetic marks in adult mouse testis have shown that the region from -800 to +900 bp respective to Cdh2 transcription start site (TSS) is likely the active regulatory region of this gene. In addition, the JASPAR 2022 matrix has predicted an AP-1 binding element at about -600 bp. Transcription factors of the activator protein 1 (AP-1) family have been implicated in the regulation of the expression of genes encoding cell-to-cell interaction proteins such as Gja1, Nectin2 and Cdh3. To test the potential regulation of Cdh2 by members of the AP-1 family, siRNAs were transfected into TM4 Sertoli cells. The knockdown of Junb led to a decrease in Cdh2 expression. ChIP-qPCR and luciferase reporter assays with site-directed mutagenesis confirmed the recruitment of Junb to several AP-1 regulatory elements in the proximal region of the Cdh2 promoter in TM4 cells. Further investigation with luciferase reporter assays showed that other AP-1 members can also activate the Cdh2 promoter albeit to a lesser extent than Junb. Taken together, these data suggest that in TM4 Sertoli cells, Junb is responsible for the regulation of Cdh2 expression which requires its recruitment to the proximal region of the Cdh2 promoter.

The transcription factors Junb and Fosl2 cooperate to regulate Cdh3 expression in 15P-1 Sertoli cells.

In Sertoli cells of the testis, cadherins (Cdh) are important cell-to-cell interaction proteins and contribute to the formation of the blood-testis barrier being essential for germ cells' protection. P-cadherin or Cdh3 is only expressed in Sertoli cells from embryonic to prepubertal development. Interestingly, the expression profile of Cdh3 correlates with that of activating protein-1 (AP-1) transcription factors during Sertoli cells development. To assess their potential implications in the regulation of Cdh3, different AP-1 transcription factors were overexpressed in 15P-1 Sertoli cells. We found that the overexpressions of Junb and Fosl2 activated Cdh3 promoter. ChIP-qPCR assay and luciferase reporter assay with 5' promoter deletions and site-directed mutagenesis confirmed the recruitment of Junb and Fosl2 to an AP-1 regulatory element at -47 bp in the proximal region of Cdh3 promoter in 15P-1 cells. These findings were further supported by histone modification markers and chromatin accessibility surrounding Cdh3 promoter in mouse testis. Moreover, the knockdowns of Junb and/or Fosl2 by siRNA decreased Cdh3 protein levels. Taken together, these data suggest that in 15P-1 Sertoli cells, the AP-1 family members Junb and Fosl2 are responsible for the regulation of Cdh3 expression, which requires the recruitment of both factors to the proximal region of the Cdh3 promoter.

Involvement of calmodulin-dependent protein kinase I in the regulation of the expression of connexin 43 in MA-10 tumor Leydig cells.

Connexin 43 (Cx43, also known as Gja1) is the most abundant testicular gap junction protein. It has a crucial role in the support of spermatogenesis by Sertoli cells in the seminiferous tubules as well as in androgen synthesis by Leydig cells. The multifunctional family of Ca2+/calmodulin-dependent protein kinases (CaMK) is composed of CaMK I, II, and IV and each can serve as a mediator of nuclear Ca2+ signals. These kinases can control gene expression by phosphorylation of key regulatory sites on transcription factors. Among these, AP-1 members cFos and cJun are interesting candidates that seem to cooperate with CaMKs to regulate Cx43 expression in Leydig cells. In this study, the Cx43 promoter region important for CaMK-dependent activation is characterized using co-transfection of plasmid reporter-constructs with different plasmids coding for CaMKs and/or AP-1 members in MA-10 Leydig cells. Here we report that the activation of Cx43 expression by cFos and cJun is increased by CaMKI. Furthermore, results from chromatin immunoprecipitation suggest that the recruitment of AP-1 family members to the proximal region of the Cx43 promoter may involve another uncharacterized AP-1 DNA regulatory element and/or protein-protein interactions with other partners. Thus, our data provide new insights into the molecular regulatory mechanisms that control mouse Cx43 transcription in testicular Leydig cells.

Classical cadherins in the testis: how are they regulated?

Cadherins (CDH) are crucial intercellular adhesion molecules, contributing to morphogenesis and creating tissue barriers by regulating cells' movement, clustering and differentiation. In the testis, classical cadherins such as CDH1, CDH2 and CDH3 are critical to gonadogenesis by promoting the migration and the subsequent clustering of primordial germ cells with somatic cells. While CDH2 is present in both Sertoli and germ cells in rodents, CDH1 is primarily detected in undifferentiated spermatogonia. As for CDH3, its expression is mainly found in germ and pre-Sertoli cells in developing gonads until the establishment of the blood-testis barrier (BTB). This barrier is made of Sertoli cells forming intercellular junctional complexes. The restructuring of the BTB allows the movement of early spermatocytes toward the apical compartment as they differentiate during a process called spermatogenesis. CDH2 is among many junctional proteins participating in this process and is regulated by several pathways. While cytokines promote the disassembly of the BTB by enhancing junctional protein endocytosis for degradation, testosterone facilitates the assembly of the BTB by increasing the recycling of endocytosed junctional proteins. Mitogen-activated protein kinases (MAPKs) are also mediators of the BTB kinetics in many chemically induced damages in the testis. In addition to regulating Sertoli cell functions, follicle stimulating hormone can also regulate the expression of CDH2. In this review, we discuss the current knowledge on regulatory mechanisms of cadherin localisation and expression in the testis.

The who, what, and how of teamwork research in medical operating rooms: A scoping review.

Despite the importance of teamwork in the operating room (OR), teamwork can often be conflated with teamwork components (e.g., communication, cooperation). We reviewed the existing literature pertaining to OR teamwork to understand which teamwork components have been assessed. Following PRISMA guidelines for scoping reviews, 4,233 peer-reviewed studies were identified using MEDLINE and Embase. Eighty-seven studies were included for synthesis and analysis. Using the episodic model of teamwork as an organizing framework, studies were grouped into the following teamwork categories: (a) transition processes (e.g., goal specification), (b) action processes (e.g., coordination), (c) interpersonal processes (e.g., conflict management), (d) emergent states (e.g., psychological safety), or (e) omnibus topics (a combination of higher-order teamwork processes). Results demonstrated that action processes were most frequently explored, followed by transition processes, omnibus topics, emergent states, and interpersonal processes. Although all studies were framed as investigations of teamwork, it is important to highlight that most explored only one or a few constructs under the overarching umbrella of teamwork. We advocate for enhanced specificity with descriptions of OR teamwork, reporting practices pertaining to interprofessional demographics and outcomes, and increased diversity in study design and surgery type to advance understanding of teamwork and its implications.

Efficacy of plasma exchange in patients with anti-MDA5 rapidly progressive interstitial lung disease.

BACKGROUND: Rapidly progressive interstitial lung disease (RP-ILD) is a frequent and severe manifestation of anti-MDA5 dermatomyositis (MDA5-DM) associated with poor outcome. The optimal treatment regimen for MDA5-DM RP-ILD is yet to be determined. Specifically, the value of adding plasma exchange (PLEX) to corticosteroids and immunosuppressants remains unclear. We aimed to evaluate the effect of PLEX on the outcome of patients with MDA5-DM RP-ILD. METHODS: This French nationwide multicentre retrospective study included all MDA5-DM RP-ILD patients from 2012 to 2021 admitted to 18 centres. The primary endpoint was one-year transplant-free survival. RESULTS: 51 patients with MDA5-DM RP-ILD (female 67%; mean age at disease onset: 51 ± 11.6 years) were included. Thirty-two (63%) patients required mechanical ventilation and twenty-five (49%) received PLEX. One-year mortality or lung transplant occurred in 63% cases after a median follow-up of 77 [38-264] days. The Cox proportional hazards multivariable model only retained mechanical ventilation but not PLEX (p = 0.7) as independent predictor of the primary endpoint. One-year transplant-free survival rates in PLEX + vs. PLEX-were 20% vs. 54% (p = 0.01), respectively. The Kaplan-Meier estimated probabilities of one-year transplant-free survival was statistically higher in PLEX-compared to PLEX + patients (p = 0.05). PLEX + compared to PLEX-patients more frequently received mechanical ventilation and immunosuppressants suggesting PLEX + patients had a more severe disease. CONCLUSION: MDA5-DM RP-ILD is associated with poor rate of one-year transplant-free survival. The use of PLEX was not associated with a better outcome albeit they were mainly given to more severe patients. While our study reports the largest series of MDA5-DM RP-ILD given PLEX, these results needs to be interpreted with caution owing the numerous selection, indication and interpretation bias. Further studies are needed to evaluate their efficacy in this setting.

Non-Markovian Feedback Control and Acausality: An Experimental Study.

Causality is an important assumption underlying nonequilibrium generalizations of the second law of thermodynamics known as fluctuation relations. We here experimentally study the nonequilibrium statistical properties of the work and of the entropy production for an optically trapped, underdamped nanoparticle continuously subjected to a time-delayed feedback control. Whereas the non-Markovian feedback depends on the past position of the particle for a forward trajectory, it depends on its future position for a time-reversed path, and is therefore acausal. In the steady-state regime, we show that the corresponding fluctuation relations in the long-time limit exhibit a clear signature of this acausality, even though the time-reversed dynamics is not physically realizable.

Basic Leucine Zipper Transcription Factors as Important Regulators of Leydig Cells' Functions.

Transcription factors members of the basic leucine zipper (bZIP) class play important roles in the regulation of genes and functions in testicular Leydig cells. Many of these factors, such as cAMP responsive element binding protein 1 (CREB1) and CCAAT enhancer binding protein beta (CEBPB), are regulated by the cAMP/protein kinase A (PKA) pathway, the main signaling pathway activated following the activation of the luteinizing hormone/choriogonadotropin membrane receptor LHCGR by the - hormone LH. Others, such as X-box binding protein 1 (XBP1) and members of the cAMP responsive element binding protein 3 (CREB3)-like superfamily, are implicated in the endoplasmic reticulum stress by regulating the unfolded protein response. In this review, the influences of bZIP transcription factors, including CREB1, CEBPB and activator protein 1 (AP-1) family members, on the regulation of genes important for cell proliferation, steroidogenesis and Leydig cell communication will be covered. In addition, unresolved questions regarding the mechanisms of actions of bZIP members in gene regulation will be identified.

Gigantol Improves Cholesterol Metabolism and Progesterone Biosynthesis in MA-10 Leydig Cells.

In aging males, androgen production by testicular Leydig cells decreases at a rate of approximately 1% per year. Phenolic compounds may enhance testosterone biosynthesis and delay the onset of male hypogonadism. Gigantol is a bibenzyl compound isolated from several types of orchids of the genus Dendrobium. This compound has various biological activities, including antioxidant activity. However, its capacity to regulate gene expression and steroid production in testicular Leydig cells has never been evaluated. We investigated the effect of gigantol on MA-10 Leydig cells' gene expression using an RNA-Seq approach. To further investigate the structure-function relationship of the hydroxy-methoxyphenyl moiety of gigantol, experiments were also performed with ferulic acid and isoferulic acid. According to transcriptomic analysis, all genes coding for cholesterol biosynthesis-related enzymes are increased in response to gigantol treatment, resulting in increased lipid droplets accumulation. Moreover, treatments with 10 µM gigantol increased StAR protein levels and progesterone production from MA-10 Leydig cells. However, neither ferulic acid nor isoferulic acid influenced StAR protein synthesis and progesterone production in MA-10 Leydig cells. Thus, our findings indicate that gigantol improves cholesterol and steroid biosynthesis within testicular Leydig cells.

Generation of optically synchronized pump-signal beams for ultrafast OPCPA via the optical Kerr effect.

In recent years, multi-petawatt laser installations have achieved unprecedented peak powers, opening new horizons to laser-matter interaction studies. Ultra-broadband and extreme temporal contrast pulse requirements make optical parametric chirped pulse amplification (OPCPA) in the few-picosecond regime the key technology in these systems. To guarantee high fidelity output, however, OPCPA requires excellent synchronization between pump and signal pulses. Here, we propose a new highly versatile architecture for the generation of optically synchronized pump-signal pairs based on the Kerr shutter effect. We obtained >550µJ pump pulses of 12 ps duration at 532 nm optically synchronized with a typical ultrashort CPA source at 800 nm. As a proof-of-principle demonstration, our system was also used for amplification of ∼20µJ ultra-broadband pulses based on an OPCPA setup.

Whole exome sequencing, a hypothesis-free approach to investigate recurrent early miscarriage.

RESEARCH QUESTION: Are there genetic determinants shared by unrelated women with unexplained recurrent early miscarriage (REM)? DESIGN: Thirty REM cases and 30 controls were selected with extreme phenotype among women from Eastern Brittany (France), previously enrolled in an incident case-control study on thrombophilic mutations. Cases and controls were selected based on the number of early miscarriages or live births, respectively. Peripheral blood was collected for DNA extraction at initial visit. The burden of low-frequency variants in the coding part of the genes was compared using whole exome sequencing (WES). RESULTS: Cases had 3 to 17 early miscarriages (20 cases: ≥5 previous losses). Controls had 1 to 4 live births (20 controls: ≥3 previous live births) and no miscarriages. WES data were available for 29 cases and 30 controls. A total of 209,387 variants were found (mean variant per patient: 59,073.05) with no difference between groups (P = 0.68). The top five most significantly associated genes were ABCA4, NFAM1, TCN2, AL078585.1 and EPS15. Previous studies suggest the involvement of vitamin B12 deficiency in REM. TCN2 encodes for vitamin B12 transporter into cells. Therefore, holotranscobalamin (active vitamin B12) was measured for both cases and controls (81.2 ± 32.1 versus 92.9 ± 34.3 pmol/l, respectively, P = 0.186). Five cases but no controls were below 50 pmol/l (P = 0.052). CONCLUSIONS: This study highlights four new genes of interest in REM, some of which belong to known networks of genes involved in embryonic development (clathrin-mediated endocytosis and ciliary pathway). The study also confirms the involvement of TCN2 (vitamin B12 pathway) in the early first trimester of pregnancy.

Transcriptome modulation following administration of luteolin to bleomycin-etoposide-cisplatin chemotherapy on rat LC540 tumor Leydig cells.

Leydig cell tumours represent 1%-3% of all cases of testicular tumours in men. Such tumours respond poorly to radiation or chemotherapy, including bleomycin-etoposide-cisplatin (BEP) combinatorial therapy. In this study, we investigated an alternative approach involving luteolin to improve the efficacy of chemotherapy. LC540 tumour Leydig cells were treated with BEP (bleomycin 40 µg/ml, etoposide 4 µg/ml, cisplatin 8 µg/ml) and/or luteolin 10 µM for comparison with DMSO-treated cells. We performed a transcriptome analysis using RNA-Seq to characterise changes in biological processes and signalling pathways. Treatments of LC540 tumour Leydig cells with luteolin significantly decreased the expression of genes involved in cholesterol biosynthesis, while increasing the expression of genes related to glutathione conjugation (p < .05). Genes being significantly upregulated in response to BEP treatment were involved in the response to toxic substances and transcriptional regulation. Oppositely, genes being significantly downregulated by BEP treatment were enriched for intracellular signal transduction, cell migration, cell adhesion, reproductive system development and cholesterol biosynthesis. BEP chemotherapy proved to be effective in increasing gene expression related to apoptosis of tumour Leydig cells. However, addition of luteolin to BEP treatment had no other effects on biological processes or pathways related to cancer treatment.

A Season-Long Examination of Team Structure and Its Implications for Subgroups in Individual Sport.

The authors explored how sport structure predisposed a team to subgroup formation and influenced athlete interactions and team functioning. A season-long qualitative case study was undertaken with a nationally ranked Canadian track and field team. Semistructured interviews were conducted with coaches (n = 4) and athletes (n = 11) from different event groups (e.g., sprinters, jumpers) at the beginning and at the end of the season. The results highlighted constraints that directly impacted athlete interactions and predisposed the group to subgroup formation (e.g., sport/event type, facility/schedule limitations, team size/change over time). The constraints led to structural divides that impacted interactions but could be overcome through team building, engaging with leaders, and prioritizing communication. These findings underline how structure imposed by the design of sports impacts teammate interactions and how practitioners, coaches, and athletes can manage groups when facing such constraints. The authors describe theoretical and practical implications while also proposing potential future directions.

"Beyond the Rink": A Multilevel Analysis of Social Identity Behaviors Captured Using the Electronically Activated Recorder.

This study used ecological sampling methods to examine associations between youth athletes' experiences receiving and engaging in behaviors indicative of in-group ties, cognitive centrality, and in-group affect (i.e., social identity) during a 3-day competitive ice hockey tournament. Forty-five youth (Mage = 12.39 years; SDage = 1.14 years; 94% male) from nine teams wore an electronically activated recorder that captured brief (50-s) audio observations throughout the tournament. Participants also completed daily diary questionnaires for each day of competition. Multilevel structural equation modeling demonstrated that athletes were more likely to engage in behaviors indicative of in-group affect and cognitive centrality on days when they received as higher-than-average frequency of behaviors indicative of cognitive centrality from teammates, coaches, and parents. The findings suggest that when team members interact in ways that demonstrate they are thinking about their team, they influence fellow members to behave in ways that promote a sense of "us."

Improvement in the temporal contrast in the tens of ps range of the multi-PW Apollon laser front-end.

We demonstrate the impact of the optics roughness in Öffner stretchers used in chirped pulse amplification laser chains and how it is possible to improve the temporal contrast ratio in the temporal range of 10-100 ps by adequately choosing the optical quality of the key components. Experimental demonstration has been realized in the front-end source of the multi-petawatt (PW) laser facility Apollon, resulting in an enhancement of the contrast ratio by two to three orders of magnitude.

Luteolin modulates gene expression related to steroidogenesis, apoptosis, and stress response in rat LC540 tumor Leydig cells.

In males, androgens are mainly produced by Leydig cells from the testis. A critical and highly regulated step of steroidogenesis involves the importation of cholesterol within the mitochondria by the steroidogenic acute regulatory (STAR) protein. During aging, STAR protein levels in Leydig cells gradually decrease, leading to a reduced entry of cholesterol into mitochondria and lower testosterone production. In addition to preserving its steroidogenic capacity, tumor Leydig cells can also be excellent models for evaluating the mechanisms of action of anticancer agents. In this study, we examined whether polyphenolics having structural similarities to luteolin could promote steroidogenic and cancer-related gene expressions within rat L540 tumor Leydig cells. In this cell model, luteolin activated Star expression and increased progesterone as well as testosterone productions. Interestingly, luteolin decreased gene expression related to cholesterol biosynthesis, possibly inhibiting membrane synthesis and cell proliferation. In addition, increased expression of genes such as Fas, Cdkn1a, Atp7b, and Tp53, as well as increased accumulation of cleaved caspase 3 and PARP, in response to luteolin treatment indicates that apoptosis is being activated. Luteolin also modulated the expression of genes involved in stress response, such as glutathione-S transferases Gsta1 and Gstt2, and the unfolded protein response. Thus, dietary luteolin may be effective in Leydig cell tumor chemoprevention and in maintaining steroidogenesis in aging males.

Shock to the Heart: Psychosocial Implications and Applications of Sudden Cardiac Death in the Young.

PURPOSE OF REVIEW: Although rare, sudden cardiac death (SCD) in the young is a tragic event, having a dramatic impact upon all involved. The psychosocial burden associated with SCD can leave friends, families, and entire communities bereft. With only limited evidence to describe the volatile emotional reactions associated with a young SCD, there is an urgent need for care providers to better understand the psychological complexities and impacts faced by both at-risk individuals and those directly affected by these tragic events. RECENT FINDINGS: Current knowledge of the psychosocial implications associated with SCD in the young has recently generated interest in the cardiovascular community, with the goal of addressing prevention strategies (screening), family bereavement, and the psychological impact of at-risk or surviving individuals. With the emergence of novel strategies aimed at reducing the public health impact of SCD in the young, further discussion regarding the psychosocial impact of SCD, encompassing prevention, survivorship, and the downstream communal effects of a young death is required. Support systems and intervention could assist in the management of the associated psychosocial burden, yet there is a lack of clinical guidelines to direct this form of care. There is an important need for multidisciplinary collaboration across subspecialties to provide support to grieving individuals and manage patient well-being throughout the screening process for SCD. This collaborative approach requires the integration of cardiovascular and psychological expertise where relevant.

Transcriptomic analysis of overexpressed SOX4 and SOX8 in TM4 Sertoli cells with emphasis on cell-to-cell interactions.

Sertoli cells are localized in seminiferous tubules within the testis. They are the first testicular cells to differentiate during male sex determination. In the adult, Sertoli cells provide nutrients to germ cells, control factors for spermatogenesis and protection by establishing the blood-testis barrier (BTB). This BTB is composed of tight junctions, basal ectoplasmic specializations, adherent junctions and gap junctions. The transcription factor SOX8 is necessary for the maintenance of spermatogenesis during adult life whereas SOX4 is involved in developmental processes. These factors are highly expressed in Sertoli cells. However, few of their target genes in adult Sertoli cells are known. Hence, we compared the transcriptomes of TM4 Sertoli cells overexpressing or not SOX4 or SOX8 using RNA-Seq followed by pathways and networks analyses. We found that SOX4 overexpression leads to downregulated genes enriched for cell junction organization and positive regulation of cell-to-cell adhesion. Upregulated genes in response to SOX8 overexpression were enriched for Sertoli cell development and differentiation. However, downregulated genes were enriched for cell-to-cell adhesion, tight junction interactions, gap junctions' assembly, as well as extracellular matrix binding. Hence, our results confirm that SOX8 is an important mediator of Sertoli cell maturation, whereas SOX4 and SOX8 influence gene expression related to regulation of blood-testis barrier assembly. In addition, TM4 cells can be considered as a useful model to better define the regulatory mechanisms of SOX4 or SOX8 on gene transcription in Sertoli cells.