High Concentration of Iron Ions Contributes to Ferroptosis-Mediated Testis Injury.

In order to explore the effect of excessive iron supplementation on ferroptosis in mouse testes, Kunming mice received injections of varying concentrations of iron. The organ weight, sperm density, and malformation rate were measured. Observations of pathological and ultrastructural alterations in spermatogenic tubules were conducted using haematoxylin eosin (HE) staining and transmission electron microscopy(TEM). Transcript levels of related genes and serum biochemical indicators were measured in mouse testicular tissue. The results showed that higher iron concentration inhibited the growth of mice; reduced the organ coefficients of the testis, heart, and liver; and increased the rate of sperm malformation and mortality. Supplementation with high levels of iron ions can adversely affect the male reproductive system by reducing sperm count, damaging the structure of the seminiferous tubules and causing sperm cell abnormalities. In addition, the iron levels also affected the immune response and blood coagulation ability by affecting the red blood cells, white blood cells and platelets. The results showed that iron ions can affect mouse testicular tissue and induce ferroptosis by altering the expression of ferroptosis-related genes. However, the degree of effect was different for the different concentrations of iron ions. The study also revealed the potential role of deferoxamine in inhibiting the occurrence of ferroptosis. Nevertheless, the damage caused to the testis by deferoxamine supplementation suggests the need for further research in this direction. This study provides reference for reproductive toxicity induced by environmental iron exposure and clarifies the mechanism of reproductive toxicity caused by iron overload and the important role of iron in the male reproductive system.

Double-Tube Multiplex TaqMan Real-Time PCR for the Detection of Eight Animal-Derived Dairy Ingredients.

Milk and dairy products represent important sources of nutrition in our daily lives. The identification of species within dairy products holds importance for monitoring food adulteration and ensuring traceability. This study presented a method that integrated double-tube and duplex real-time polymerase chain reaction (PCR) with multiplex TaqMan probes to enable the high-throughput detection of animal-derived ingredients in milk and dairy products. The detection system utilized one pair of universal primers, two pairs of specific primers, and eight animal-derived specific probes for cow, buffalo, goat, sheep, camel, yak, horse, and donkey. These components were optimized within a double-tube and four-probe PCR multiplex system. The developed double-tube detection system could simultaneously identify the above eight targets with a detection limit of 10-0.1 pg/µL. Validation using simulated adulterated milk samples demonstrated a detection limit of 0.1%. The primary advantage of this method lies in the simplification of the multiplex quantitative real-time PCR (qPCR) system through the use of universal primers. This method provides an efficient approach for detecting ingredients in dairy products, providing powerful technical support for market supervision.

Isolation and Identification of Non-Saccharomyces Yeast Producing 2-Phenylethanol and Study of the Ehrlich Pathway and Shikimate Pathway.

2-phenylethanol (2-PE) has been widely utilized as an aromatic additive in various industries, including cosmetics, beer, olive oil, tea, and coffee, due to its rose-honey-like aroma. However, no reports have investigated the production of 2-PE by Starmerella bacillaris. Here, S. bacillaris (syn., Candida zemplinina, and named strain R5) was identified by analysis of morphology, physiology and biochemistry, and 26S rRNA and ITS gene sequence. Then, based on the analysis of whole-genome sequencing and comparison with the KEGG database, it was inferred that strain R5 could synthesize 2-PE from L-phe or glucose through the Ehrlich pathway or shikimate pathway. For further verification of the 2-PE synthesis pathway, strain R5 was cultured in M3 (NH4+), M3 (NH4+ + Phe), and M3 (Phe) medium. In M3 (Phe) medium, the maximum concentration of 2-PE reached 1.28 g/L, which was 16-fold and 2.29-fold higher than that in M3 (NH4+) and M3 (Phe + NH4+) media, respectively. These results indicated that 2-PE could be synthesized by strain R5 through the shikimate pathway or Ehrlich pathway, and the biotransformation from L-phe to 2-PE was more efficient than that from glucose. The qRT-PCR results suggested that compared to M3 (Phe + NH4+) medium, the mRNA expression levels of YAT were 124-fold and 86-fold higher in M3 (Phe) and M3 (NH4+) media, respectively, indicating that the transport of L-phe was inhibited when both NH4+ and Phe were present in the medium. In the M3 (Phe) and M3 (Phe + NH4+) media, the mRNA expression level of ADH5 was higher than PDC, hisC, GOT1, and YAT, and it was 2.6 times higher and 2.48 times higher, respectively, compared to the M3 (NH4+) medium, revealing that the key gene catalyzing the dehydrogenation of benzaldehyde to 2-PE is ADH5. Furthermore, strain R5 exhibits tolerance to high concentrations of 2-PE, reaching 3 g/L, which conferred an ideal tolerance to 2-PE. In summary, the synthesis pathway of 2-PE, mainly for the Ehrlich pathway, was proved for the first time in S. bacillaris, which had not been previously explored and provided a basis for non-Saccharomyces yeast-producing 2-PE and its applications.

Species Identification of Caviar Based on Multiple DNA Barcoding.

This study aimed to explore the applicability of DNA barcoding for assessing the authenticity of caviar on the Chinese market. A set of universal COI primers and two sets of designed primers based on COI and D-loop genes were used to identify maternal species of samples from 21 batches of caviar. The results showed that the PCR products from three sets of primers had more than 98% similarity to the sequences in database. The COI gene could not distinguish sturgeons with closed genetic relationships, while D-loop gene could effectively improve the accuracy of DNA barcoding and was more suitable to the identification of interspecific sturgeon than the COI gene. The neighbor-joining dendrogram further confirmed the applicability and accuracy of COI and D-loop genes in identifying maternal relatives of caviar (Acipenser baerii/Acipenser gueldenstaedtii/Acipenser schrenckii/Huso dauricus/Huso huso). Despite the limitations of mitochondrial DNA in identifying hybrid sturgeon species, the presence of counterfeit caviar of non-sturgeon ingredients could be excluded. All the caviar samples were identified successfully as sturgeon species, but the mislabeling rate of species was 33.4%, indicating that there were illegal phenomena such as disorderly labeling, mislabeling, and adulteration on the market.

Evolutionary and reverse engineering in Saccharomyces cerevisiae reveals a Pdr1p mutation-dependent mechanism for 2-phenylethanol tolerance.

BACKGROUND: 2-Phenylethanol (2-PE), a higher alcohol with a rose-like odor, inhibits growth of the producer strains. However, the limited knowledge regarding 2-PE tolerance mechanisms renders our current knowledge base insufficient to inform rational design. RESULTS: To improve the growth phenotype of Saccharomyces cerevisiae under a high 2-PE concentration, adaptive laboratory evolution (ALE) was used to generate an evolved 19-2 strain. Under 2-PE stress, its OD600 and growth rate increased by 86% and 22% than that of the parental strain, respectively. Through whole genome sequencing and reverse engineering, transcription factor Pdr1p mutation (C862R) was revealed as one of the main causes for increased 2-PE tolerance. Under 2-PE stress condition, Pdr1p mutation increased unsaturated fatty acid/saturated fatty acid ratio by 42%, and decreased cell membrane damage by 81%. Using STRING website, we identified Pdr1p interacted with some proteins, which were associated with intracellular ergosterol content, reactive oxygen species (ROS), and the ATP-binding cassette transporter. Also, the results of transcriptional analysis of genes encoded these proteins confirmed that Pdr1p mutation induced the expression of these genes. Compared with those of the reference strain, the ergosterol content of the PDR1_862 strain increased by 72%-101%, and the intracellular ROS concentration decreased by 38% under 2-PE stress. Furthermore, the Pdr1p mutation also increased the production of 2-PE (11% higher). CONCLUSIONS: In the present work, we have demonstrated the use of ALE as a powerful tool to improve yeast tolerance to 2-PE. Based on the reverse engineering, transcriptional and physiological analysis, we concluded that Pdr1p mutation significantly enhanced the 2-PE tolerance of yeast by regulating the fatty acid proportion, intracellular ergosterol and ROS. It provides new insights on Pdr1p mediated 2-PE tolerance, which could help in the design of more robust yeasts for natural 2-PE synthesis.

Rapamycin enhanced the production of 2-phenylethanol during whole-cell bioconversion by yeast.

2-Phenylethanol (2-PE), a higher alcohol with a rose-like odor, has been widely utilized in food, perfume, and beverages. Saccharomyces cerevisiae is one of the most promising microorganisms for the biosynthesis of natural 2-PE. However, the growth of S. cerevisiae is generally inhibited by 2-PE, which makes its production in yeast cell factories challenging. Here, the whole-cell bioconversion was used to avert growth inhibition, leading to an increase in the concentration and productivity of 2-PE. Moreover, rapamycin (Rap) addition further improved the efficiency of 2-PE synthesis. The concentration of 2-PE (2.20 g/L) was 1.68-fold higher than that in the absence of Rap during the whole-cell bioconversion by S. cerevisiae BY4741. RT-qPCR results showed that Rap addition increased the transcription of ARO9, ARO10, ADH2, GAP1, ARO80, GLN3, and GDH2. When the GLN3 was knocked out, the transcriptional levels of the genes were dramatically decreased, and the concentration of 2-PE significantly decreased to 0.21 g/L. The results indicated that Rap enhanced the flux of the Ehrlich pathway, and Gln3 exerted a central role in the regulation of Rap. Furthermore, commercial yeast (S. cerevisiae FY202001) was selected to verify the applicability of Rap. In the presence of Rap, 3.67 g/L 2-PE was obtained by whole-cell bioconversion in flask, which was increased by 9% than that in the absence of Rap. Finally, the 2-PE titer reached 4.93 g/L by whole-cell bioconversion in a 5 L bioreactor, with a yield of 84 mol% from L-phenylalanine and a productivity of 0.103 g/L h, which was far higher than that of the currently reported in S. cerevisiae. These findings provided a new idea for the efficient synthesis of 2-PE. KEY POINTS: • Whole-cell bioconversion was used to produce 2-PE. • The regulation of the Ehrlich pathway by Rap provides a theoretical basis for developing an effective yeast cell factory to produce 2-PE. • The 2-PE productivity of 0.103 g/L h is far higher than that of the currently reported in S. cerevisiae .

Differential analysis of ergosterol function in response to high salt and sugar stress in Zygosaccharomyces rouxii.

Zygosaccharomyces rouxii is an osmotolerant and halotolerant yeast that can participate in fermentation. To understand the mechanisms of salt and sugar tolerance, the transcription levels of Z. rouxii M 2013310 under 180 g/L NaCl stress and 600 g/L glucose stress were measured. The transcriptome analysis showed that 2227 differentially expressed genes (DEGs) were identified under 180 g/L NaCl stress, 1530 DEGs were identified under 600 g/L glucose stress, and 1278 DEGs were identified under both stress conditions. Then, KEGG enrichment analyses of these genes indicated that 53.3% of the upregulated genes were involved in the ergosterol synthesis pathway. Subsequently, quantitative PCR was used to verify the results, which showed that the genes of the ergosterol synthesis pathway were significantly upregulated under 180 g/L NaCl stress. Finally, further quantitative testing of ergosterol and spotting assays revealed that Z. rouxii M 2013310 increased the amount of ergosterol in response to high salt stress. These results highlighted the functional differences in ergosterol under sugar stress and salt stress, which contributes to our understanding of the tolerance mechanisms of salt and sugar in Z. rouxii.

Sensing, Uptake and Catabolism of L-Phenylalanine During 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in Saccharomyces cerevisiae.

2-Phenylethanol (2-PE) is an important flavouring ingredient with a persistent rose-like odour, and it has been widely utilized in food, perfume, beverages, and medicine. Due to the potential existence of toxic byproducts in 2-PE resulting from chemical synthesis, the demand for "natural" 2-PE through biotransformation is increasing. L-Phenylalanine (L-Phe) is used as the precursor for the biosynthesis of 2-PE through the Ehrlich pathway by Saccharomyces cerevisiae. The regulation of L-Phe metabolism in S. cerevisiae is complicated and elaborate. We reviewed current progress on the signal transduction pathways of L-Phe sensing, uptake of extracellular L-Phe and 2-PE synthesis from L-Phe through the Ehrlich pathway. Moreover, the anticipated bottlenecks and future research directions for S. cerevisiae biosynthesis of 2-PE are discussed.

Zygosaccharomyces rouxii, an Aromatic Yeast Isolated From Chili Sauce, Is Able to Biosynthesize 2-Phenylethanol via the Shikimate or Ehrlich Pathways.

We isolated an aromatic strain of yeast (M2013310) from chili sauce. Assembly, annotation, and phylogenetic analysis based on genome sequencing, identified M2013310 as an allodiploid yeast that was closely related to Zygosaccharomyces rouxii. During fermentation, M2013310, produced an aromatic alcohol with a rose-honey scent; gas chromatography tandem mass spectrometry identified this alcohol as 2-phenylethanol. The concentration of 2-phenylethanol reached 3.8 mg/L, 1.79 g/L, and 3.58 g/L, in M3 (NH4 +), M3 (NH4 + + Phe), and M3 (Phe) culture media, after 72 h of fermentation, respectively. The mRNA expression levels of ARO8 encoding aromatic aminotransferases I and ARO10 encoding phenylpyruvate decarboxylase by M2013310 in M3 (Phe) were the lowest of the three different forms of media tested. These results indicated that M2013310 can synthesize 2-phenylethanol via the Shikimate or Ehrlich pathways and the production of 2-phenylethanol may be significantly improved by the over-expression of these two genes. Our research identified a promising strain of yeast (M2013310) that could be used to improve the production of 2-phenylethanol.

Telomere dysfunction promotes genome instability and metastatic potential in a K-ras p53 mouse model of lung cancer.

Current mouse models of lung cancer recapitulate signature genetic lesions and some phenotypic features of human lung cancer. However, because mice have long telomeres, models to date do not recapitulate the aspects of lung carcinogenesis-telomere attrition and the genomic instability that ensues-believed to serve as key mechanisms driving lung tumor initiation and progression. To explore the contributions of telomere dysfunction to lung cancer progression, we combined a telomerase catalytic subunit (mTerc) mutation with the well-characterized K-rasG12D mouse lung cancer model. K-ras(G12D) mTerc(-/-) mice with telomere dysfunction but intact p53 exhibited increased lung epithelial apoptosis, delayed tumor formation and increased life span relative to K-ras(G12D) mTerc(+/-) mice with intact telomere function. This demonstrates that by itself, telomere dysfunction acts in a tumor-suppressive mechanism. Introduction of a heterozygous p53 mutation exerted a marked histopathological, biological and genomic impact. K-ras(G12D) mTerc(-/-) p53(+/-) mice developed aggressive tumors with more chromosomal instabilities and high metastatic potential, leading to decreased overall survival. Thus, we have generated a murine model that more faithfully recapitulates key aspects of the human disease. Furthermore, these findings clearly demonstrate (in an in vivo model system) the dual nature of telomere shortening as both a tumor-suppressive and tumor-promoting mechanism in lung cancer, dependent on p53 status.

Photocatalytic degradation of Acid Blue 62 over CuO-SnO2 nanocomposite photocatalyst under simulated sunlight.

The novel CuO-SnO2 nanocomposite oxide photocatalysts were prepared by simple co-precipitation method, and characterized by X-ray diffraction, transmission electron microscopy, N2 adsorption-desorption measurement and UV-Vis diffuse reflectance spectroscopy. The photocatalytic activities of CuO-SnO2, evaluated using the photodegradation of Acid Blue 62 as a probe reaction under the irradiation of Xenon light, were also found to be related to the calcination temperature and the molar ratio of Cu to Sn. The maximum photocatalytic activity of the CuO-SnO2 photocatalyst was observed to be calcined at 500 degrees C for 3 h (the molar ratio of Cu to Sn was 1:1) due to the sample with good crystallization and high surface area. It also showed much higher photocatalytic activity in treatment dye wastewater under simulated sunlight irradiation compared to Degussa P25 TiO2.

[Shengdi injection on rat model of lung inflammation induced by lipopolysaccharides].

OBJECTIVE: To investigate the effect of Shengdi injection on rat model of lung inflammation. METHOD: The rat model was established by intratrachea instillation of lipopolysaccharides (LPS). The total and different white blood cell counts in bronchoalvoelar lavage fluid (BALF) were performed and the level of tumor necrosis factor-alpha (TNF-alpha), superoxide anion radical (O2-) and myeloperoxidase (MPO) was measured, as well as pathologic change of pulmonary tissue was tested. RESULT: Shengdi injection could depress the increasing of the amount of total white blood cells and neutrophils and inhibit the increasing of TNF-alpha, O2-, MPO caused by LPS, as well as relieve the pathologic change including Neutrophils infiltrating and mucous edema in tracheae after intravenous administration. While it did not show the effect on monocyte, and histological lesion of the lung tissue. CONCLUSION: Shengdi injection shows some anti-inflammatory effect in rat lung induced by LPS and it can be concluded tentatively that anti-inflammatory, inhibiting the release of cytokine and inflammatory medium, and antioxidation are some of the mechanism of its effect on COPD.

DNA-dependent protein kinase catalytic subunit is not required for dysfunctional telomere fusion and checkpoint response in the telomerase-deficient mouse.

Telomeres are key structural elements for the protection and maintenance of linear chromosomes, and they function to prevent recognition of chromosomal ends as DNA double-stranded breaks. Loss of telomere capping function brought about by telomerase deficiency and gradual erosion of telomere ends or by experimental disruption of higher-order telomere structure culminates in the fusion of defective telomeres and/or the activation of DNA damage checkpoints. Previous work has implicated the nonhomologous end-joining (NHEJ) DNA repair pathway as a critical mediator of these biological processes. Here, employing the telomerase-deficient mouse model, we tested whether the NHEJ component DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was required for fusion of eroded/dysfunctional telomere ends and the telomere checkpoint responses. In late-generation mTerc(-/-) DNA-PKcs(-/-) cells and tissues, chromosomal end-to-end fusions and anaphase bridges were readily evident. Notably, nullizygosity for DNA Ligase4 (Lig4)--an additional crucial NHEJ component--was also permissive for chromosome fusions in mTerc(-/-) cells, indicating that, in contrast to results seen with experimental disruption of telomere structure, telomere dysfunction in the context of gradual telomere erosion can engage additional DNA repair pathways. Furthermore, we found that DNA-PKcs deficiency does not reduce apoptosis, tissue atrophy, or p53 activation in late-generation mTerc(-/-) tissues but rather moderately exacerbates germ cell apoptosis and testicular degeneration. Thus, our studies indicate that the NHEJ components, DNA-PKcs and Lig4, are not required for fusion of critically shortened telomeric ends and that DNA-PKcs is not required for sensing and executing the telomere checkpoint response, findings consistent with the consensus view of the limited role of DNA-PKcs in DNA damage signaling in general.

Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors.

The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation.