Characterization of the transcriptional responses of Armillaria gallica 012m to GA3.

Gastrodia elata needs to establish a symbiotic relationship with Armillaria strains to obtain nutrients and energy. However, the signaling cross talk between G. elata and Armillaria strains is still unclear. During our experiment, we found that the vegetative mycelium of Armillaria gallica 012m grew significantly better in the media containing gibberellic acid (GA3) than the blank control group (BK). To explore the response mechanism, we performed an RNA-sequencing experiment to profile the transcriptome changes of A. gallica 012m cultured in the medium with exogenous GA3. The transcriptome-guided differential expression genes (DEGs) analysis of GA3 and BK showed that a total of 1309 genes were differentially expressed, including 361 upregulated genes and 948 downregulated genes. Some of those DEGs correlated with the biological process, including positive regulation of chromosome segregation, mitotic metaphase/anaphase transition, attachment of mitotic spindle microtubules to kinetochore, mitotic cytokinesis, and nuclear division. These analyses explained that GA3 actively promoted the growth of A. gallica to some extent. Further analysis of protein domain features showed that the deduced polypeptide contained 41 candidate genes of GA receptor, and 27 of them were expressed in our samples. We speculate that GA receptors exist in A. gallica 012m. Comparative studies of proteins showed that the postulated GA receptor domains of A. gallica 012m have a higher homologous correlation with fungi than others based on cluster analysis.

MiR-195-5p is involved in testicular ischemia/reperfusion injury by directly targeting PELP1 and regulating spermatogonia pyroptosis.

BACKGROUND AND OBJECTIVE: Ischemia/reperfusion injury (IRI), which is characterized by testicular torsion and causes permanent impairment of spermatogenic function, is linked with pyroptosis. Studies have implicated endogenous small non-coding RNAs in IRI development across various organs. In this study, we elucidated the mechanism underlying miR-195-5p's action in regulating pyroptosis in testicular IRI. METHODS: We established two models, namely a testicular torsion/ detorsion (T/D) mouse model and an oxygen-glucose deprivation/reperfusion (OGD/R)-treated germ cell model. Hematoxylin and eosin staining was performed to evaluate the testicular ischemic injury. The expression of pyroptosis-related proteins and reactive oxygen species production in testis tissues were detected using Western blotting, quantitative real-time PCR, malondialdehyde and superoxide dismutase assay kits and immunohistochemistry. Cell viability and cytotoxicity were evaluated using CCK-8 and LDH assays, whereas expression patterns of inflammatory proteins were measured using ELISA, immunofluorescence, and western blot assays. miR-195-5p interaction with PELP1 was validated by conducting the luciferase enzyme reporter test. RESULTS: Pyroptosis-related proteins NLRP3, GSDMD, IL-1ß, and IL-18 were significantly upregulated following testicular IRI. A similar pattern was observed in the OGD/R model. miR-195-5p was significantly downregulated in mouse IRI testis tissue and OGD/R-treated GC-1 cells. Notably, miR-195-5p downregulation promoted whereas its upregulation attenuated pyroptosis in OGD/R-treated GC-1 cells. Furthermore, we found that PELP1 is a miR-195-5p target. miR-195-5p attenuated pyroptosis in GC-1 cells by inhibiting PELP1 expression during OGD/R, and this protective effect was blocked upon miR-195-5p downregulation. Collectively, these results indicated that miR-195-5p inhibits testicular IRI-induced pyroptosis by targeting PELP1, suggesting that it has the potential to serve as a novel target for the future development of therapies for testicular torsion.

Comparative transcriptome analysis of Armillaria gallica 012m in response to ethephon treatment.

Background: Gastrodia elata, known as a rootless, leafless, achlorophyllous and fully mycoheterotrophic orchid, needs to establish symbionts with particular Armillaria species to acquire nutrition and energy. Previous research findings had approved that ethylene (ET) played an important role in plant-fungi interaction and some receptors of ET had been discovered in microorganisms. However, the molecular mechanisms underlying the role of ET in the interaction between G. elata and Armillaria species remain unknown. Methods: Exiguous ethephon (ETH) was added to agar and liquid media to observe the morphological features of mycelium and count the biomass respectively. Mycelium cultured in liquid media with exiguous ETH (0.1 ppm, 2.0 ppm, 5.0 ppm) were chosen to perform whole-transcriptome profiling through the RNA-seq technology (Illumina NGS sequencing). The DEGs of growth-related genes and candidate ET receptor domains were predicted on SMART. Results: ETH-0.1 ppm and ETH-2 ppm could significantly improve the mycelium growth of A. gallica 012m, while ETH-5 ppm inhibited the mycelium growth in both solid and liquid media. The number of up-regulated or down-regulated genes increased along with the concentrations of ETH. The growth of mycelia might benefit from the up-regulated expression of Pyr_redox (Pyridine nucleotide-disulphide oxidoreductase), GAL4 (C6 zinc finger) and HMG (High Mobility Group) genes in the ETH-0.1 ppm and ETH-2 ppm. Therefore, the growth of mycelia might be impaired by the down-regulated expression of ZnF_C2H2 and ribosomal protein S4 proteins in the ETH-5 ppm. Seven ET receptor domains were predicted in A. gallica 012m. Based on cluster analysis and comparative studies of proteins, the putative ETH receptor domains of A. gallica 012m have a higher homologous correlation with fungi. Conclusions: The responses of A. gallica 012m to ETH had a concentration effect similar to the plants' responses to ET. Therefore, the number of up-regulated or down-regulated genes are increased along with the concentrations of ETH. Seven ET receptor protein domains were predicted in the genome and transcriptome of A. gallica 012m. We speculate that ETH receptors exist in A. gallica 012m and ethylene might play an important role in the plant-fungi interaction.

Emodin alleviates testicular ischemia-reperfusion injury through the inhibition of NLRP3-mediated pyroptosis.

Ischemia-reperfusion injury (IRI) is a major cause of injury after testicular torsion and can lead to permanent impairment of spermatogenesis. Emodin (6-methyl-1,3,8-trihydroxyanthraquinone) has potent anti-inflammatory effects and may be protective against IRI in various organs. Herein, we evaluated the effects of emodin on pyroptosis in spermatogenic cells and its role in the process of testicular IRI. A testicular torsion/detorsion (TTD) mouse model and an oxygen-glucose deprivation/reperfusion (OGD/R) germ cell model were established. Hematoxylin and eosin staining was performed to evaluate the testicular ischemic injury. The expression of pyroptosis-related proteins and reactive oxygen species production in testis tissues were detected using Western blotting, quantitative real-time PCR, malondialdehyde and superoxide dismutase assay kits and immunohistochemistry. Cell viability and cytotoxicity were evaluated using Cell Counting Kit-8 and lactate dehydrogenase assay kit. Enzyme-linked immunosorbent assay, immunofluorescence and immunoblotting were performed to assess inflammatory protein levels. The results revealed that pyroptosis and inflammation levels were upregulated after testicular IRI, and emodin inhibited inflammation and pyroptosis by acting on NOD-like receptor thermal protein domain-associated protein 3 (NLRP3). Emodin exerts protective effects on testicular IRI by acting on the NLRP3 signaling pathway and inhibiting IRI-mediated pyroptosis. Emodin treatment attenuated testicular IRI and inhibited pyroptosis. Inhibitory effects of emodin on pyroptosis were attributed to the inhibition of NLRP3 inflammasomes. Thus, emodin could be an alternative treatment for testicular IRI.

Transcriptome analysis of Armillaria gallica 012 m in response to auxin.

Gastrodia elata is an achlorophyllous and fully mycoheterotrophic orchid which obtains carbon and other nutrients from Armillaria species in its life cycle. Many researchers suggested that plant hormones, as signing molecules, play a central role in the plant-fungi interaction. In the process of Armillaria gallica 012 m cultivation, both exogenous indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) distinctly stimulated the growth of mycelia in solid media. The differential expression genes (DEGs) of A. gallica 012 m with IAA versus blank control (BK) and IBA versus BK were investigated. The results showed that more than 80% of DEGs of the IAA group were coincident with the DEGs of the IBA group, and more than half of upregulated DEGs and most of the downregulated DEGs of the IAA group coincided with those DEGs of the IBA group. Above research implied that A. gallica 012 m could perceive IAA and IBA, and possess similar responses and signaling pathways to IAA and IBA. The overlapping differential genes of the IAA group and IBA group were analyzed by GO term, and the results showed that several DEGs identified were related to biological processes including positive regulation of the biological process and biological process. The downregulated NmrA-like and FKBP_C genes might be benefit to the growth of mycelia. Those results can explain that exiguous IAA and IBA improved the growth of A. gallica to some extent. We speculate that IAA and IBA are signaling molecules, and regulate the expression of growth-related genes of A. gallica 012 m by the same signaling pathway.

Evaluation of aliphatic acid metabolism in bladder cancer with the goal of guiding therapeutic treatment.

Urothelial bladder cancer (BLCA) is a common internal malignancy with a poor prognosis. The re-programming of lipid metabolism is necessary for cancer cell growth, proliferation, angiogenesis and invasion. However, the role of aliphatic acid metabolism genes in bladder cancer patients has not been explored. The samples' gene expression and clinicopathological data were obtained from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Univariate, multivariate, and LASSO Cox regression were used to develop a BLCA prognostic model. GSVA was used to assess function, whereas pRRophetic was used to assess chemotherapeutic drug sensitivity. The twelve-gene signature may define the tumor immune milieu, according to the risk score model. We compared the expression of aliphatic acid metabolism genes in malignant and non-cancerous tissues and chose 90 with a false discovery rate of 0.05 for The Cancer Genome Atlas cohort. The prognostic risk score model can effectively predict BLCA OS. A nomogram including age, clinical T stage, gender, grade, pathological stage, and clinical M stage was developed as an independent BLCA prognostic predictor. The halfmaximal inhibitory concentration (IC50) was used to assess chemotherapeutic medication response. Sorafenib and Pyrimethamine were used to treat patients with low risk scores more sensitively than patients with high risk scores. Immunotherapy candidates with CMS1 exhibited higher risk ratings. The aliphatic acid prognostic risk score model can assess metabolic trends. Clinical stage and molecular subtype may be used to categorize individuals using the risk score.With this new paradigm, future cancer treatment and immunotherapy may be tailored to the patient's exact requirements.

Oleuropein attenuates testicular ischemia-reperfusion by inhibiting apoptosis and inflammation.

BACKGROUND: Ischemia-reperfusion injury (IRI) is the key reason of injury after testicular torsion and may eventually lead to male infertility. Oleuropein, a natural antioxidant isolated from Olea europaea, has shown beneficial effects in different models of ischemia. We evaluated the effects of oleuropein on testicular IRI and explored the underlying protective mechanisms. METHODS: A mouse testicular torsion/detorsion (T/D) model and an oxygen-glucose deprivation/reperfusion (OGD/R) germ cell model were established and treated with oleuropein. H&E staining was used to evaluate testicular pathological changes. Apoptosis and apoptosis-associated protein levels in testis tissues were assessed by TUNEL staining, immunohistochemical staining and western blot. Apoptosis levels and apoptosis-associated protein levels in GC-1 were evaluated by flow cytometry, immunofluorescence and western blot. Oxidative stress levels were assessed by malondialdehyde (MDA) and superoxide dismutase (SOD) kits. Cell viability and inflammatory protein levels were evaluated by CCK-8 assay coupled with qRT-PCR. RESULTS: Relative to the control group, SOD activity was markedly suppressed, while MDA, Bax, Caspase-3, TNF-α as well as IL-1ß levels were significantly increased in the T/D model and OGD/R model. However, all of the aforementioned alterations were relieved by oleuropein treatment. CONCLUSION: Our findings indicate that oleuropein may be a promising treatment option to attenuate testicular IRI via its anti-oxidant, anti-inflammatory as well as anti-apoptotic properties.

Long Non-coding RNA MEG3 Promotes Pyroptosis in Testicular Ischemia-Reperfusion Injury by Targeting MiR-29a to Modulate PTEN Expression.

Increasing evidence shows that the abnormal long non-coding RNAs (lncRNAs) expression is closely related to ischemia-reperfusion injury (I/R) progression. Studies have previously described that lncRNA MEG3 regulates pyroptosis in various organs I/R. Nevertheless, the related mechanisms of MEG3 in testicular I/R has not been clarified. The aim of this research is to unravel underlying mechanisms of the regulation of pyroptosis mediated by MEG3 during testicular I/R. We have established a testicular torsion/detorsion (T/D) model and an oxygen-glucose deprivation/reperfusion (OGD/R)-treated spermatogenic cell model. Testicular ischemic injury was assessed by H&E staining. Western blotting, quantitative real-time PCR, MDA, and SOD tests and immunohistochemistry measured the expression of MEG3 and related proteins and the level of ROS production in testicular tissues. Quantitative real-time PCR and western blotting determined the relative expression of MEG3, miR-29a, and relevant proteins in GC-1. Cell viability and cytotoxicity were measured by CCK-8 and LDH assays. Secretion and expression levels of inflammatory proteins were determined by ELISA, immunofluorescence and western blotting. The interaction among MEG3, miR-29a, and PTEN was validated through a dual luciferase reporter assay and Ago2-RIP. In this research, we identified that MEG3 was upregulated in animal specimens and GC-1. In loss of function or gain of function assays, we verified that MEG3 could promote pyroptosis. Furthermore, we found that MEG3 negatively regulated miR-29a expression at the posttranscriptional level and promoted PTEN expression, and further promoted pyroptosis. Therefore, we explored the interaction among MEG3, miR-29a and PTEN and found that MEG3 directly targeted miR-29a, and miR-29a targeted PTEN. Overexpression of miR-29a effectively eliminated the upregulation of PTEN induced by MEG3, indicating that MEG3 regulates PTEN expression by targeting miR-29a. In summary, our research indicates that MEG3 contributes to pyroptosis by regulating miR-29a and PTEN during testicular I/R, indicating that MEG3 may be a potential therapeutic target in testicular torsion.

[Novel coronavirus induces testicular injury: Analysis of causes and follow-up monitoring].

The outbreak of coronavirus disease 2019 (COVID-19) caused by 2019 novel coronavirus has become a global public health challenge. In addition to the typical respiratory symptoms, COVID-19 can induce damage to testicular spermatogenesis. This study focuses on the possible causes and follow-up monitoring of testicular injury induced by COVID-19.