A Novel Cuprotosis-Related Gene FDX1 Signature for Overall Survival Prediction in Clear Cell Renal Cell Carcinoma Patients.

Background: Ferredoxin 1 (FDX1) is a newly discovered gene regulating cuprotosis. However, the effect of FDX1 expression on clear renal cell carcinoma (ccRCC) is unknown. Methods: Gene expression profiles and clinical data of ccRCC patients were downloaded from the Cancer Genome Atlas (TCGA) database. The differences in FDX1 expression between ccRCC and nonneoplastic tissues adjacent to cancer were analyzed by R software. The results were validated by GEO data, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting (WB), and immunohistochemistry (IHC). Chi-square test was used to analyze the clinical pathological parameters. Kaplan-Meier survival analysis and Cox proportional hazard regression model selection were used to evaluate the effect of FDX1 expression on overall survival. Protein interaction networks were used to analyze other proteins that interact with FDX1. Signal pathway analysis was performed for possible FDX1 enrichment using GSEA and ssGSEA algorithms. Pan-cancer analysis of FDX1 was carried out through TCGA database. Results: The FDX1 expression in nontumor tissues was significantly higher than that in ccRCC, and the expression difference was verified by GEO data, qRT-PCR, WB, and IHC. The high expression of FDX1 was significantly related to the well overall survival rate (P < 0.05). The chi-square test showed that the high expression of FDX1 was related to gender, TNM stage, T stage, lymph node metastasis, and pathological grade. Additionally, the FDX1 expression level was different in groups classified based on pathological grade, gender, TNM stage, T stage, lymph node metastasis, and distant metastasis (P < 0.05). The multivariate analysis revealed the high expression of FDX1 as an important independent predictor for overall survival. STRING database results showed that LIAS and LIPT1 may interact with FDX1 in the PPI network, which are also involved in the regulation of cuprotosis. The GSEA and ssGSEA results showed that the FDX1 was enriched in the anticancer pathway. The FDX1 high expression is associated with better prognosis in many cancers, as revealed by pan-cancer analysis. Conclusion: FDX1 may play a role in the progression of ccRCC as a tumor suppressor gene. It can be used as a potential prognostic indicator and therapeutic target of ccRCC. However, the cuprotosis regulatory role in the development of ccRCC needs to be further verified.

Association of PD-1/PD-L1 expression and Epstein--Barr virus infection in patients with invasive breast cancer.

PURPOSE: Causative factors of breast cancer include infections, such as Epstein-Barr virus (EBV) infection. The aim of this study was to analyze the clinicopathological features of EBV-positive (IBC) and determine if EBV affects programmed cell death receptor 1 (PD-1)/PD ligand 1 (PD-L1) expression in IBC, similar to other EBV-infected tumors with PD-L1/PD-1 expression. METHODS: We collected 140 samples of IBC tissues and 25 samples of adjacent tissues. All patients were followed-up by telephone from the day of surgery to December 2020. Chromogenic in-situ hybridization was performed to evaluate EBV-encoded RNA (EBER). Immunohistochemistry was performed to evaluate PD-L1 and PD-1 expressions. The correlation between PD1/PDL1 expression and clinicopathological features was also analyzed. RESULTS: EBER was detected in 57 of 140 (40.7%) IBC tissues and not detected in any adjacent tissue (P < 0.05). Clinicopathologic features of patients were consistent with EBV-associated IBC. EBV infection was correlated with the mass size, menopausal status, axillary lymph node metastasis, vascular invasion, Ki-67 index, clinical stage, and estrogen receptor and progesterone receptor expressions (all P < 0.05), but not with the histological type, invasive ductal carcinoma histological grade, or human epidermal growth factor receptor 2 (HER2) expression (all P > 0.05). The positive rate of PD-1/PD-L1 expression was higher in the EBV-positive group than in the EBV-negative group (P < 0.05). The Kaplan-Meier univariate survival analysis showed that EBV was associated with poor disease-free survival and overall survival in patients with IBC. PD-L1/PD-1 expression could predict a poor prognosis. CONCLUSIONS: In this study, clinicopathologic characteristics of patients were consistent with EBV-infected IBC. Patients with EBV-positive breast cancer were more likely to have elevated PD-1/PDL-1 expression compared to those with EBV-negative breast cancer. This finding could serve as a basis to explore therapeutic targets, particularly immunotherapy, for patients with IBC.

Diagnosis of an extremely rare case of malignant adenomyoepithelioma in pleomorphic adenoma: A case report.

BACKGROUND: Pleomorphic adenoma (PA) is the most common type of salivary gland tumor, and its common sites are parotid gland, sinus, nasal septum and cleft palate. PA is an uncommon benign type of tumor occurring in the breast, and there are few reports of cases in Asia. CASE SUMMARY: An 84-year-old woman found a mass in the upper outer quadrant of the right breast > 1 year ago. The patient underwent a right breast lumpectomy and sentinel lymph node biopsy. The pathological diagnosis was PA in the upper outer quadrant of the right breast, and the malignant component was malignant adenomyoepithelioma. The postoperative course was uneventful, and no chemotherapy was administered. At 18 mo of follow-up, the patient is alive and well, with no evidence of recurrent disease. CONCLUSION: Patients with breast PA should first undergo extended excision of breast masses followed by pathological examination. If malignancy is confirmed or the surgical margin is positive, modified radical mastectomy should be performed.

PHLDA3 inhibition attenuates endoplasmic reticulum stress-induced apoptosis in myocardial hypoxia/reoxygenation injury by activating the PI3K/AKT signaling pathway.

Endoplasmic reticulum stress (ERS)-induced apoptosis serves a crucial role in the pathogenesis of myocardial ischemia/reperfusion injury (MIRI). Previous studies have confirmed that pleckstrin homology-like domain family A member 3 (PHLDA3) is an important mediator in ERS-associated apoptosis. The aim of the current study focused on whether PHLDA3 served protective effects on hypoxia/reoxygenation (H/R)-injured cardiomyocytes by inhibiting ERS-induced apoptosis. Furthermore, the molecular mechanisms associated with the PI3K/AKT signaling pathway were investigated. Primary neonatal rat cardiomyocytes were isolated and randomized into four groups: i) Control + adenovirus encoding scrambled short hairpin RNA (AdshRNA); ii) control + adenoviral vectors encoding PHLDA3 shRNA (AdshPHLDA3); iii) H/R+ AdshRNA and iv) H/R+AdshPHLDA3. AdshPHLDA3 was used to knock down PHLDA3. An H/R injury model was constructed by treatment with hypoxia for 4 h followed by reoxygenation for 6 h. A PI3K/AKT inhibitor, LY294002, was supplemented in mechanistic studies. Cell viability and LDH/CK releases were detected to evaluate myocardial damage. Flow cytometry assays were used to assess apoptotic response. Western blotting assays were used to detect protein expression. The results demonstrated that H/R induced myocardial damage and increased PHLDA3 expression. ERS-induced apoptosis was significantly increased following H/R injury, as indicated by increased apoptotic rates and ERS-associated protein expression, including those of CHOP, 78 kDa glucose-regulated protein and caspase-12. However, PHLDA3 inhibition following AdshPHLDA3 transfection reversed cell damage and ERS-associated apoptosis on H/R injury. Studies for molecular mechanisms concluded that the apoptosis-inhibition effects and cardioprotective roles of PHLDA3 inhibition were induced partly by the activation of the PI3K/AKT pathway, which was verified by LY294002 treatment. In conclusion, in the process of H/R injury, PHLDA3 inhibition reduced ERS-induced apoptosis and H/R injury by activating the PI3K/AKT pathway. PHLDA3 may be a therapeutic target for the treatment of MIRI.

Using a Heat Diffusion Model to Detect Potential Drug Resistance Genes of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the oldest known and most dangerous diseases. Although the spread of TB was controlled in the early 20th century using antibiotics and vaccines, TB has again become a threat because of increased drug resistance. There is still a lack of effective treatment regimens for a person who is already infected with multidrug-resistant Mtb (MDR-Mtb) or extensively drug-resistant Mtb (XDRMtb). In the past decades, many research groups have explored the drug resistance profiles of Mtb based on sequence data by GWAS, which identified some mutations that were significantly linked with drug resistance, and attempted to explain the resistance mechanisms. However, they mainly focused on several significant mutations in drug targets (e.g. rpoB, katG). Some genes which are potentially associated with drug resistance may be overlooked by the GWAS analysis. OBJECTIVE: In this article, our motivation is to detect potential drug resistance genes of Mtb using a heat diffusion model. METHODS: All sequencing data, which contained 127 samples of Mtb, i.e. 34 ethambutol-, 65 isoniazid-, 53 rifampicin- and 45 streptomycin-resistant strains. The raw sequence data were preprocessed using Trimmomatic software and aligned to the Mtb H37Rv reference genome using Bowtie2. From the resulting alignments, SAMtools and VarScan were used to filter sequences and call SNPs. The GWAS was performed by the PLINK package to obtain the significant SNPs, which were mapped to genes. The P-values of genes calculated by GWAS were transferred into a heat vector. The heat vector and the Mtb protein-protein interactions (PPI) derived from the STRING database were inputted into the heat diffusion model to obtain significant subnetworks by HotNet2. Finally, the most significant (P < 0.05) subnetworks associated with different phenotypes were obtained. To verify the change of binding energy between the drug and target before and after mutation, the method of molecular dynamics simulation was performed using the AMBER software. RESULTS: We identified significant subnetworks in rifampicin-resistant samples. Excitingly, we found rpoB and rpoC, which are drug targets of rifampicin. From the protein structure of rpoB, the mutation location was extremely close to the drug binding site, with a distance of only 3.97 Å. Molecular dynamics simulation revealed that the binding energy of rpoB and rifampicin decreased after D435V mutation. To a large extent, this mutation can influence the affinity of drug-target binding. In addition, topA and pyrG were reported to be linked with drug resistance, and might be new TB drug targets. Other genes that have not yet been reported are worth further study. CONCLUSION: Using a heat diffusion model in combination with GWAS results and protein-protein interactions, the significantly mutated subnetworks in rifampicin-resistant samples were found. The subnetwork not only contained the known targets of rifampicin (rpoB, rpoC), but also included topA and pyrG, which are potentially associated with drug resistance. Together, these results offer deeper insights into drug resistance of Mtb, and provides potential drug targets for finding new antituberculosis drugs.

Development of a Molecular Marker for Fruiting Body Pattern in Auricularia auricula-judae.

The fruiting body pattern is an important agronomic trait of the edible fungus Auricularia auricula-judae, and an important breeding target. There are two types of fruiting body pattern: the cluster type and the chrysanthemum type. We identified the fruiting body pattern of 26 test strains, and then constructed two different near-isogenic pools. Then, we developed sequence characterized amplified region (SCAR) molecular markers associated with the fruiting body pattern based on sequence-related amplified polymorphism (SRAP) markers. Ten different bands (189-522 bp) were amplified using 153 pairs of SRAP primers. The SCAR marker "SCL-18" consisted of a single 522-bp band amplified from the cluster-type strains, but not the chrysanthemum strains. This SCAR marker was closely associated with the cluster-type fruiting body trait of A. auricula-judae. These results lay the foundation for further research to locate and clone genes controlling the fruiting body pattern of A. auricula-judae.

AGC1.5 Kinase Phosphorylates RopGEFs to Control Pollen Tube Growth.

Double fertilization in angiosperms requires the targeted delivery of immotile sperm to the eggs through pollen tubes. The polarity of tip-growing pollen tubes is maintained through dynamic association of active Rho GTPases of plants (ROP-GTP) with the apical plasma membrane. Guanine nucleotide exchange factors for ROPs (RopGEFs) catalyze the activation of ROPs and thereby affect spatiotemporal ROP signaling. Whereas RopGEFs have been found to be phosphorylated proteins, the kinases responsible for their phosphorylation in vivo and biological consequences of RopGEF phosphorylation in pollen tube growth remain unclear. We report here that the Arabidopsis AGC1.5 subfamily of cytoplasmic kinases is critical for the restricted localization of ROP-GTP during pollen tube growth. Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of active ROPs and defective events downstream of ROP signaling in pollen tubes. AGC1.5 interacts with RopGEFs via their catalytic PRONE domain and phosphorylates RopGEFs at a conserved Ser residue of PRONE domain. Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of RopGEFs in pollen tubes, similar to the phenotype caused by the mutation that renders RopGEFs non-phosphorylatable by AGC1.5. Collectively, our results provide mechanistic insights into the spatiotemporal activation of ROPs during the polar growth of pollen tubes.

Arabidopsis VAC14 Is Critical for Pollen Development through Mediating Vacuolar Organization.

Pollen viability depends on dynamic vacuolar changes during pollen development involving increases and decreases of vacuolar volume through water and osmolite accumulation and vacuolar fission. Mutations in FAB1A to FAB1D, the genes encoding phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]-converting kinases, are male gametophyte lethal in Arabidopsis (Arabidopsis thaliana) due to defective vacuolar fission after pollen mitosis I, suggesting a key role of the phospholipid in dynamic vacuolar organization. However, other genetic components that regulate the production of PI(3,5)P2 and its involvement in pollen germination and tube growth are unknown. Here, we identified and characterized Arabidopsis VAC14, a homolog of the yeast and metazoan VAC14s that are crucial for the production of PI(3,5)P2VAC14 is constitutively expressed and highly present in developing pollen. Loss of function of VAC14 was male gametophyte lethal due to defective pollen development. Ultrastructural studies showed that vacuolar fission after pollen mitosis I was compromised in vac14 mutant microspores, which led to pollen abortion. We further showed that inhibiting the production of PI(3,5)P2 or exogenous application of PI(3,5)P2 mimicked or rescued the pollen developmental defect of the vac14 mutant, respectively. Genetic interference and pharmacological approaches suggested a role of PI(3,5)P2 in pollen germination and tube growth. Our results provide insights into the function of VAC14 and, by inference, that of PI(3,5)P2 in plant cells.

Construction of a genetic linkage map and QTL mapping of agronomic traits in Auricularia auricula-judae.

Auricularia auricula-judae is a traditional edible fungus that is cultivated widely in China. In this study, a genetic linkage map for A. auricula-judae was constructed using a mapping population consisting of 138 monokaryons derived from a hybrid strain (A119-5). The monokaryotic parent strains A14-5 and A18-119 were derived from two cultivated varieties, A14 (Qihei No. 1) and A18 (Qihei No. 2), respectively. In total, 130 simple sequence repeat markers were mapped. These markers were developed using the whole genome sequence of A. auricula-judae and amplified in A14-5, A18- 119, and the mapping population. The map consisted of 11 linkage groups (LGs) spanning 854 cM, with an average interval length of 6.57 cM. A testcross population was derived from crossing between the monokaryon A184-57 (from the wild strain A184 as a tester strain) and the mapping population. Important agronomic trait-related QTLs, including mycelium growth rate on potato dextrose agar for the mapping population, mycelium growth rate on potato dextrose agar and sawdust for the testcross population, growth period (days from inoculation to fruiting body harvesting), and yield for the testcross population, were identified using the composite interval mapping method. Six mycelium growth raterelated QTLs were identified on LG1 and LG4, two growth period-related QTLs were identified on LG2, and three yieldrelated QTLs were identified on LG2 and LG6. The results showed no linkage relationship between mycelium growth rate and growth period. The present study provides a foundation for locating genes for important agronomic characteristics in A. auricula-judae in the future.

[Effect of Acorus tatarinowii Schott on the amino acid neurotransmitters in the striatum focal cerebral ischemia in rat].

OBJECTIVE: To research the change of concentration of the amino acid neurotransmitters in the striatum focal cerebral ischemia in rat and the effect of Acorus tatarinowii Schott, one of inducing resuscitation drugs, for 4 of amino acid neurotransmitters. METHODS: Twenty four rats were divided into four groups (n = 6): control group, cerebral ischemia group, sham operation group and Acorns tatarinowii Schott treated group. Rats were established into models of cerebral ischemia by occluding bilateral thread cork method. Formation sampling were performed in a striatum area using microdialysis and the detection of biological sample including aspartic acid, glutamic acid, glycine and gamma-aminobutyric acid by high performance liquid chromatography (HPLC) electrochemical detector system. RESULTS: Compared with the control, the all contents of 4 kinds of the amino acids were significantly increased during cerebral ischemia (P < 0.01). Compared with the cerebral ischemia group, the contents of aspartic acid, glutamic acid that were excitatory amino acids were remarkably decreased in the striatum for Acorus tatarinowii Schott treated group (P < 0.01), It was no significant influence on gamma-aminobutyric acid and glycine that belonged to inhibitory amino acid in a nascent condition but with a elevating in the later period of microdialysis. CONCLUSION: Acorus tatarinowii Schott can enter the cerebral parenchyma through blood brain barrier and cut down glutamic acid,aspartic acid increased during cerebral ischemia. As a result, the neurotoxicity attributed to the excitatory amino acid has been released in excessive amounts declined so as to avoid the secondary impairment of neurons caused by excitatory amino acids pernicious effects after ischemia. It may be one of the protective mechanism of drugs for inducing resuscitation resembling EAA receptor antagonists to ischemi brain.

[Di-2-ethylhexyl phthalate and its metabolite single-ethylhexyl phthalate affect TGF-beta 1 expression and telomerase activity in the testis of young male rats].

OBJECTIVE: To investigate the influences of di-2-ethylhexyl phthalate (DEHP) and its metabolite single-ethylhexyl phthalate (MEHP) on the expression of transforming growth factor-beta 1 (TGF-beta1) and telomerase activity in young male Wistar METHODS: Ninety-six 2-week-old male Wistar rats were equally randomized into a normal control (NC) group, a positive control (PC) group, and six experimental groups. Those of the NC group were intragastrically administered 0.9% normal saline at a dose of 0.2 ml per kg per d for 3 weeks, those in the PC group cyclophosphamide (CTX) at 100 mg per kg per d for 1 week, and those of the experimental groups DEHP and MEHP, respectively, at a low dose (100 mg per kg per d) for 3 weeks, a moderate dose (200 mg per kg per d) for 2 weeks, and a high dose (300 mg per kg per d) for 1 week. Then we observed the morphological changes of the testicular sperm and counted the sperm heads and their abnormity rate at different doses and times. We detected the expression of TGF-beta1 in the testis tissue using immunohistochemical SABC and RT-PCR, measured the area density, and determined telomerase activity by ELISA. RESULTS: Compared with the NC group, the experimental groups showed an obvious reduction in the total sperm count and number of sperm heads (P < 0.05) and a significant increase in the rate of teratosperm (P < 0.05), such as decapitated, hookless, and double-tailed sperm. And there were no significant differences between the high-dose short-term and low-dose long-term medication groups (P > 0.05). The expression of TGF-beta1 was low in the NC group, high in the PC group, and obviously increased in the membrane and cytoplasm of spermatogenic cells of the experimental groups. The area density and TGF-beta1 mRNA expression were 0.156 0 +/- 0.003 5 and 1.51 +/- 0.20 in the NC group, 0.534 0 +/- 0.003 1 and 8.43 +/- 1.75 in the PC group, 0.289 0 +/- 0.003 6 and 3.83 +/- 1.57 in the DEHP groups, and 0.284 0 +/- 0.003 1 and 3.51 +/- 1.41 in the MEHP groups. There were significant differences between the experimental and the other two groups (P < 0.01), but not between the high-dose short-term and low-dose long-term medication groups (P > 0.05). Telomerase activity was remarkably reduced in the experimental groups as compared with the NC group (P < 0.05), but with no significant difference between the high-dose short-term and low-dose long-term medication groups (P > 0.05). CONCLUSION: DEHP and its metabolite MEHP can evidently induce spermatogenic injury in young male rats, which may be associated with their induction of increased TGF-beta1 expression and decreased telomerase activity in the rat testis.