Nontargeted Identification of Organic Components in Fine Particulate Matter Related to Lung Tumor Metastasis Based on an Adverse Outcome Pathway Strategy.

Emerging studies implicate fine particulate matter (PM2.5) and its organic components (OCs) as urgent hazard factors for lung cancer progression in nonsmokers. Establishing the adverse outcome pathway (AOP)-directed nontargeted identification method, this study aimed to explore whether PM2.5 exposure in coal-burning areas promoted lung tumor metastasis and how we identify its effective OCs to support traceability and control of regional PM2.5 pollution. First, we used a nude mouse model of lung cancer for PM2.5 exposure and found that the exposure significantly promoted the hematogenous metastases of A549-Luc cells in lung tissues and the adverse outcomes (AOs), with key events (KEs) including the changed expression of epithelial-mesenchymal transition (EMT) markers, such as suppression of E-cad and increased expression of Fib. Subsequently, using AOs and KEs as adverse outcome directors, we identified a total of 35 candidate chemicals based on the in vitro model and nontargeted analysis. Among them, tributyl phosphate (C12H27O4P), 2-bromotetradecane (C14H29Br), and methyl decanoate (C11H22O2) made greater contributions to the AOs. Finally, we clarified the interactions between these OCs and EMT-activating transcription factors (EMT-ATFs) as the molecular initiation event (MIE) to support the feasibility of the above identification strategy. The present study updates a new framework for identifying tumor metastasis-promoting OCs in PM2.5 and provides solid data for screening out chemicals that need priority control in polluted areas posing higher lung cancer risk.

NAMPT encapsulated by extracellular vesicles from young adipose-derived mesenchymal stem cells treated tendinopathy in a "One-Stone-Two-Birds" manner.

BACKGROUND: Tendinopathy is the leading sports-related injury and will cause severe weakness and tenderness. Effective therapy for tendinopathy remains limited, and extracellular vesicles (EVs) derived from adipose tissue-derived mesenchymal stem cells (ADMSCs) have demonstrated great potential in tendinopathy treatment; however, the influence of aging status on EV treatment has not been previously described. RESULTS: In this study, it was found that ADMSCs derived from old mice (ADMSCold) demonstrated remarkable cellular senescence and impaired NAD+ metabolism compared with ADMSCs derived from young mice (ADMSCyoung). Lower NAMPT contents were detected in both ADMSCold and its secreted EVs (ADMSCold-EVs). Advanced animal experiments demonstrated that ADMSCyoung-EVs, but not ADMSCold-EVs, alleviated the pathological structural, functional and biomechanical properties in tendinopathy mice. Mechanistic analyses demonstrated that ADMSCyoung-EVs improved cell viability and relieved cellular senescence of tenocytes through the NAMPT/SIRT1/PPARγ/PGC-1α pathway. ADMSCyoung-EVs, but not ADMSCold-EVs, promoted phagocytosis and M2 polarization in macrophages through the NAMPT/SIRT1/Nf-κb p65/NLRP3 pathway. The macrophage/tenocyte crosstalk in tendinopathy was influenced by ADMSCyoung-EV treatment and thus it demonstrated "One-Stone-Two-Birds" effects in tendinopathy treatment. CONCLUSIONS: This study demonstrates an effective novel therapy for tendinopathy and uncovers the influence of donor age on curative effects by clarifying the detailed biological mechanism.

Reversible protection of Cys-93(ß) by PEG alters the structural and functional properties of the PEGylated hemoglobin.

As a potential hemoglobin (Hb)-based oxygen carrier (HBOC), the PEGylated Hb has received much attention for its non-nephrotoxicity. However, PEGylation can adversely alter the structural and functional properties of Hb. The site of PEGylation is an important factor to determine the structure and function of the PEGylated Hb. Thus, protection of some sensitive residues of Hb from PEGylation is of great significance to develop the PEGylated Hb as HBOC. Here, Cys-93(ß) of Hb was conjugated with 20 kDa polyethylene glycol (PEG20K) through hydrazone and disulfide bonds. Then, the conjugate was modified with PEG5K succinimidyl carbonate (PEG5K-SC) using acylation chemistry, followed by removal of PEG20K Hb with hydrazone hydrolysis and disulfide reduction. Reversible conjugation of PEG20K at Cys-93(ß) can protect Lys-95(ß), Val-1(α) and Lys-16(α) of Hb from PEGylation with PEG5K-SC. The autoxidation rate, oxygen affinity, structural perturbation and tetramer instability of the PEGylated Hb were significantly decreased upon protection with PEG20K. The present study is expected to improve the efficacy of the PEGylated Hb as an oxygen therapeutic.

Wnt/ß-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components.

Wnt signaling is an evolutionarily conserved pathway that regulates cell proliferation, differentiation and apoptosis. To investigate the possible role of Wnt signaling in the regulation of ovarian follicular development, secondary follicles were isolated and cultured in vitro in the presence or absence of its activator (LiCl or Wnt3a) or inhibitor (IWR-1). We have demonstrated that activation of ß-catenin signals by activators dramatically suppressed follicular development by increasing granulosa cell apoptosis and inhibiting follicle steroidogenesis. In contrast, inhibition of Wnt signaling by IWR-1 was observed with better developed follicles and increased steroidogenesis. Further studies have shown that the transcription factor Forkhead box O3a (Foxo3a) and its downstream target molecules were modulated by the activators or the inhibitor. These findings provide evidence that Wnt signaling might negatively regulate follicular development potentially through Foxo3a signaling components.

The Heat-Induced Reversible Change in the Blood-Testis Barrier (BTB) Is Regulated by the Androgen Receptor (AR) via the Partitioning-Defective Protein (Par) Polarity Complex in the Mouse.

Scrotal hypothermia is essential for normal spermatogenesis, and temporal heat stress causes a reversible disruption of the blood-testis barrier (BTB). Previous studies have shown that AR expression in primary monkey Sertoli cells (SCs) was dramatically reduced after temporary heat treatment. However, the mechanisms underlying the heat-induced reversible disruption of the BTB, including whether it is directly regulated by the AR, remain largely unknown. In this study, we demonstrated that the AR acts upstream to regulate the heat-induced reversible change in the BTB in mice. When the AR was overexpressed in SCs using an adenovirus, the heat stress-induced down-regulation of BTB-associated proteins (Zonula occludens-1 (ZO-1), N-Cadherin, E-Cadherin, α-Catenin, and ß-Catenin) was partially rescued. AR knockdown by RNAi or treatment with flutamide (an AR antagonist) in SCs inhibited the recovery of BTB-associated protein expression after 43°C heat treatment for 30 min. The results of an in vivo AR antagonist injection experiment further showed that the recovery of BTB permeability induced by temporal heat stress was regulated by the AR. Furthermore, we observed that the co-localization and interactions of partitioning-defective protein (Par) 6-Par3-aPKC-Cdc42 polarity complex components were disrupted in both AR-knockdown and heat-induced SCs. AR overexpression in SCs prevented the disruption of these protein-protein interactions after heat treatment. AR knockdown or treatment with flutamide in SCs inhibited the restoration of these protein-protein interactions after heat treatment compared with heat treatment alone. Together, these results demonstrate that the AR plays a crucial role in the heat-induced reversible change in BTB via the Par polarity complex.

The Wilms tumor gene, Wt1, maintains testicular cord integrity by regulating the expression of Col4a1 and Col4a2.

Wt1 is specifically expressed in Sertoli cells in the developing testis. A previous study has demonstrated that Wt1 plays a critical role in maintaining the integrity of testicular cords. However, the underlying mechanism is unclear. In this study, we found that the laminin-positive basal lamina lining the testicular cords was fragmented and completely absent in some areas of Wt1(-/flox); Amh-Cre testes, indicating that the testicular cord disruption can be attributed to the breakdown of the basement membrane. To explore the molecular mechanism underlying this effect, we examined the expression of cell adhesion molecules (CAMs) and testicular cord basal lamina components by real-time RT-PCR, Western blotting, and immunostaining. Compared with control testes, the expression of CAMs (such as E-cadherin, N-cadherin, claudin11, occludin, beta-catenin, and ZO-1) was not obviously altered in Wt1(-/flox); Amh-Cre testes. However, the mRNA level of Col4a1 and Col4a2 was significantly decreased in Wt1-deficient testes. Immunostaining assays further confirmed that the collagen IV protein levels were dramatically reduced in Wt1(-/flox); Amh-Cre testes. Moreover, luciferase and point mutation analyses revealed that the Col4a1 and Col4a2 promoters were additively transactivated by WT1 and SOX9. Given this finding and previous results showing that SOX9 expression declines rapidly after Wt1 deletion, we conclude that the loss of Wt1 in Sertoli cells results in the downregulation of the important basal lamina component, which in turn causes the breakdown of the basal lamina and subsequent testicular cord disruption.

Expression of inhibin-alpha is regulated synergistically by Wilms' tumor gene 1 (Wt1) and steroidogenic factor-1 (Sf1) in sertoli cells.

Wt1 encodes a zinc finger nuclear transcriptional factor, which is specifically expressed in testicular Sertoli cells and knockdown of Wt1 in Sertoli cells causes male mice subfertility. However, the underlying mechanism is still unclear. In this study, we found that expression of inhibin-α is significantly reduced in Wt1-deficient Sertoli cells. Luciferase assays using the inhibin-α promoter indicated that the inhibin-α promoter is transactivated by the Wt1 A, and B isoforms (-KTS), but not the C, and D isoforms (+KTS). Analysis of the Wt1 responsive element of the inhibin-α promoter region using site-directed mutagenesis showed that the nucleotides between -58 and -49 are essential for Wt1-dependent transactivation of the inhibin-α promoter. ChIP assays indicated that Wt1 directly interacts with the inhibin-α promoter. In addition, the inhibin-α promoter is activated synergistically by Wt1 and Sf1. Mutation of the ligand binding domain (LBD) of Sf1 (residues 235-238) completely abolished the synergistic action between Wt1 and Sf1, but did not affect the physical interaction between these two proteins, suggesting that other factor(s) may also be involved in the regulation of inhibin-α in Sertoli cells. Further studies demonstrated that ß-catenin enhances the synergistic activation of Wt1 and Sf1 on the inhibin-α promoter. Given the fact that inhibin-α, a subunit of inhibin, is known to be involved in the regulation of spermatogenesis and testicular steroidogenesis, this study reveals a new regulatory mechanism of inhibin-α in Sertoli cells and also sheds light on the physiological functions of Wt1 in gonad development and spermatogenesis.

Testosterone induces redistribution of forkhead box-3a and down-regulation of growth and differentiation factor 9 messenger ribonucleic acid expression at early stage of mouse folliculogenesis.

Increasing evidence has shown that excess androgen may be a main cause of polycystic ovary syndrome (PCOS). However, the molecular mechanism of androgen action on the ovary is unclear. To investigate the possible impacts of androgen on early follicular development, neonatal mouse ovaries mainly containing primordial follicles were cultured with testosterone. We demonstrated that the number of primary follicles was increased after 10 d culture with testosterone treatment via phosphatidylinositol 3-kinase/Akt pathway. Androgen induced Forkhead box (Foxo)-3a activation, and translocation of Foxo3a protein from oocyte nuclei to cytoplasm, which might be a key step for primordial follicle activation. Interestingly, testosterone was also capable of down-regulating growth and differentiation factor-9 expression via its receptor. In summary, we infer that intraovarian excess androgen in PCOS might result in excess early follicles by inducing oocyte Foxo3a translocation and follicular arrest by down-regulating growth and differentiation factor-9 expression.