L-Theanine Prolongs the Lifespan by Activating Multiple Molecular Pathways in Ultraviolet C-Exposed Caenorhabditis elegans.

L-theanine, a unique non-protein amino acid, is an important bioactive component of green tea. Previous studies have shown that L-theanine has many potent health benefits, such as anti-anxiety effects, regulation of the immune response, relaxing neural tension, and reducing oxidative damage. However, little is known concerning whether L-theanine can improve the clearance of mitochondrial DNA (mtDNA) damage in organisms. Here, we reported that L-theanine treatment increased ATP production and improved mitochondrial morphology to extend the lifespan of UVC-exposed nematodes. Mechanistic investigations showed that L-theanine treatment enhanced the removal of mtDNA damage and extended lifespan by activating autophagy, mitophagy, mitochondrial dynamics, and mitochondrial unfolded protein response (UPRmt) in UVC-exposed nematodes. In addition, L-theanine treatment also upregulated the expression of genes related to mitochondrial energy metabolism in UVC-exposed nematodes. Our study provides a theoretical basis for the possibility that tea drinking may prevent mitochondrial-related diseases.

Glutathione-depletion reinforced enzyme catalytic activity for photothermal assisted bacterial killing by hollow mesoporous CuO.

The emergence and prevalence of drug-resistant bacteria caused by the overuse of antibiotics pose new challenges to the treatment of bacterial infections. In this work, hollow mesoporous CuO nanozymes (HM-CuO nanozymes) as excellent antibacterial agents were prepared by a template method. The synthesized HM-CuO nanozymes exhibit peroxidase-like catalytic activity, which can efficiently catalyze H2O2 to generate toxic reactive oxygen species (ROS), causing fatal damage to bacteria. Moreover, the hyperthermia of HM-CuO produced by photothermal therapy (PTT) not only effectively kills bacteria but also enhances the catalytic activity of nanozymes and produces more ROS. Moreover, the HM-CuO nanozymes have a glutathione (GSH)-depleting function to effectively consume GSH in bacteria and generate Cu(I) with higher catalytic effect, which can significantly improve the sterilization effect and produce a 100% inhibitory rate against E. coli and S. aureus. Overall, the HM-CuO nanozymes with strong peroxidase-like catalytic activity, excellent photothermal performance and GSH consumption ability offer a promising synergistic strategy for clinical bacterial infection.

Atorvastatin regulates vascular smooth muscle cell phenotypic transformation by epigenetically modulating contractile proteins and mediating Akt/FOXO4 axis.

Atherosclerosis (AS) is a prevalent cardiovascular disease with severe morbidity and high mortality. Phenotypic regulation of vascular smooth muscle cells (VSMCs) from the contractile and quiescent phenotype to the synthetic type is a critical step for the vascular remodeling of AS. Atorvastatin, as a 3­hydroxy­3­methyl­glutaryl coenzyme A reductase inhibitor, presents an anti­inflammatory effect to improve vascular endothelial functions. The aim of the present study was to examine the effect of atorvastatin on VSMCs phenotypic transformation and the underlying mechanism. The rat primary VSMCs were isolated and identified. The protein expression of contractile proteins, such as α­SMA, SM­MHC, and SM22α, was reduced by angiotensin II (AngII) and enhanced by atorvastatin, in which atorvastatin could reverse the effect of AngII in the VSMCs. The treatment of HDAC inhibitor trichostatin A was able to enhance AngII­inhibited expression of α­SMA and SM­MHC. Atorvastatin regulated AngII­associated VSMCs phenotypic transformation by epigenetically regulating contractile proteins. Moreover, atorvastatin modulated platelet­derived growth factor­BB (PDGF­BB)­induced VSMC phenotypic transformation by modulating the Akt/forkhead Box O4 (FOXO4) axis. Immunofluorescence analysis revealed that PDGF­BB enhanced the accumulation of FOXO4 in the VSMCs, while the treatment of atorvastatin was able to attenuate this effect and the co­treatment of Akt inhibitor LY294002 could further inhibit the phenotype. The treatment of PDGF­BB enhanced the interaction of SRF with FOXO4 and myocardin in the VSMCs, in which the co­treatment of atorvastatin and LY294002 could reverse the effect of PDGF­BB in the system. Thus, atorvastatin regulates VSMCs phenotypic transformation by epigenetically modulating contractile proteins and mediating the Akt/FOXO4 axis. Findings of the present study provide new insights into the mechanism by which atorvastatin modulates VSMCs, providing valuable evidence for the application of atorvastatin in the treatment of AS.

Cold atmospheric plasma: A non-negligible strategy for viral RNA inactivation to prevent SARS-CoV-2 environmental transmission.

Cold atmospheric plasma (CAP), regarded as a powerful physics technology, displays antimicrobial, antitumor, and even antiviral properties, but the underlying mechanism is rarely studied. In this study, four CAP exposure doses (30, 60, 120, and 240 s) were applied to inactivate a severe acute respiratory syndrome coronavirus 2 like pseudovirus on a stainless steel disk, which comprised spike protein on its membrane and can express a green fluorescent protein. In order to unravel the potential effects of CAP irradiation on pseudovirus, infection assay, optical emission spectra analysis, transmission electron microscopy (TEM), sodium dodecyl sulfate polyacrylamide gel electrophoresis, ELISA, and qPCR experiments were carried out. As a result, our study indicated that CAP irradiation can significantly decrease the infectivity of pseudovirus in a dose dependent manner through destroying the cell membrane and further damaging viral RNA, with the molecular weight and conformation of spike receptor binding domain protein unchanged.

MicroRNA-22 regulates the proliferation, drug sensitivity and metastasis of human glioma cells by targeting SNAIL1.

The Editors of JBUON issue an Expression of Concern to 'MicroRNA-22 regulates the proliferation, drug sensitivity and metastasis of human glioma cells by targeting SNAIL1', by Yunqiang Zhang, Lijun Tu, Xiuhong Zhou, Bin Li; JBUON 2020;25(1):491-496; PMID: 32277674. Following the publication of the above article, readers drew to our attention that part of the data was possibly unreliable. We sent emails to the authors with a request to provide the raw data to prove the originality, but received no reply. Therefore, as we continue to work through the issues raised, we advise readers to interpret the information presented in the article with due caution. We thank the readers for bringing this matter to our attention. We apologize for any inconvenience it may cause.

GPR37 promotes the malignancy of lung adenocarcinoma via TGF-ß/Smad pathway.

This paper aimed to research the function and in-depth mechanism of GPR37 in lung adenocarcinoma (LUAD). Herein, based on TCGA and Oncomine databases, we revealed that GPR37 was expressed at high levels in LUAD, and upregulation of GPR37 was related to the poor outcomes. Furthermore, biological function experiments in vitro were utilized to assess whether GPR37 impacts malignant phenotype of LUAD cells. Gain- or loss-of-function assays indicated that the upregulation of GPR37 contributed to improving the proliferation, migration, and invasion of LUAD cells in vitro, while knockdown of GPR37 can inhibit the malignant biological behaviors. Then, we found that depletion of GPR37 resulted in a decrease in the expression of TGF-ß1 as well as the extents of Smad2 and Smad3 phosphorylation, while overexpression of GPR37 presented opposite outcomes. Altogether, our findings indicated that GPR37 is a potential oncogene of LUAD, and its promoting effects on the malignant progression of LUAD may be realized via TGF-ß/Smad pathway.

Ganoderic acid D induces synergistic autophagic cell death except for apoptosis in ESCC cells.

ETHNOPHAMACOLOGICAL RELEVANCE: Ganoderma lucidum has been used as a medicinal mushroom for more than 2000 years in China. Ganoderic acid D (GAD) as a representative active triterpenoid from Ganoderma lucidum is known to possess anticancer activity. However, the mechanism involved in its anticancer cell process is still largely elusive. AIM OF THE STUDY: Our study aimed to investigate the anticancer effects of GAD on the esophageal squamous cell carcinoma (ESCC) cells and the underlying mechanisms at the cell level. MATERIALS AND METHODS: EC9706 and Eca109 cells were treated with GAD (0, 10, 20, 40 µM) for 24 h. The cell viability, cell cycle, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis rate, caspase-3 activity, autophagic flux, lysosomal function were examined. Cell cycle, apoptotic, autophagy and mTOR signal pathway related proteins such as P53, Cyclin B1, CytoC, PARP, Beclin-1, P62, LC3, PI3K, AKT and mTOR were analyzed by Western blot approach. RESULTS: GAD inhibited cell proliferation and induced both apoptosis and autophagic cell death. In particular, we found that in the early stage of the autophagic process, GAD could initiate and enhance the autophagy signal while in the late stage it on the contrary could block the autophagic flux by impairing the autophagosome-lysosome fusion and inhibited the lysosomal degradation. Besides the autophagic cell death, GAD also induced the apoptosis mediated by caspase-related process in parallel. The mechanism involved for the synergistic apoptotic and autophagic cell death was also explored. We found that GAD down-regulated the expression of PI3K, AKT and mTOR phosphorylated proteins in the mTOR signaling pathway which thus led to the synergistic effect on apoptosis and autophagic cell death in the ESCC cells. CONCLUSIONS: In summary, this study has documented that GAD may inhibit cell proliferation through the mTOR pathway in ESCC cells, and induce synergistic apoptosis and autophagic cell death by disrupting the autophagic flux. This work therefore also suggests that GAD may be used as an efficient anticancer adjuvant for ESCC cancer therapy.

MicroRNA-22 regulates the proliferation, drug sensitivity and metastasis of human glioma cells by targeting SNAIL1.

PURPOSE: Gliomas are aggressive brain tumors accounting for significant mortality across the globe. Biomarkers for early detection and therapeutic targets for efficient treatment are lacking for glioma. This study was undertaken to investigate the role and therapeutic implications of miR-22 in glioma. METHODS: U-87 glioma cell line was used in this study. qRT-PCR was employed for expression analysis. MTT assay was used for determination of cell viability. Lipofectamine 2000 was used for transfection. Flow cytometry was used for cell analysis. Wound healing assay and transwell assay were used for monitoring cell migration and invasion. Western blot analysis was used for estimation of protein expression. RESULTS: The miR-22 expression was found decreased in glioma cells. Overexpression of miR-22 resulted in arrest of the U-87 glioma cells at G2/M checkpoint of the cell cycle. The percentage of apoptotic U-87 cells in G2/M phase were 13.05% in negative control (NC) and 29.06% in miR-22 mimics transfected cells. The cell cycle arrest promoted by miR-22 overexpression was also associated with depletion of cyclin B1 expression in U-87 cells. Furthermore, miR-22 could also significantly increase the sensitivity of glioma U-87 cells to cisplatin. The TargetScan analysis and dual luciferase assay showed SNAIL1 to be the target of miR-22. The expression of SNAIL1 was also enhanced in all the glioma cells and miR-22 overexpression could cause suppression of the SNAIL1 expression in U-87 cells. Furthermore, SNAIL1 silencing could also cause decline in the viability of the U-87 cells. The wound healing assay showed that miR-5 overexpression caused decrease in the migration of U-87 cells, while the transwell assay showed decline in the invasion of miR-22 mimics transfected U-87 cells. CONCLUSION: Taken together, miR-22 may exhibit therapeutic implications in glioma and may prove useful in glioma treatment.

ybjX mutation regulated avian pathogenic Escherichia coli pathogenicity though stress-resistance pathway.

ybjX gene mutation decreased the pathogenicity of the avian pathogenic Escherichia coli strain, AE17. However, the associated regulatory mechanism of ybjX remains unknown. In this study, we examined the bactericidal activity of chicken serum and blood, as well as bacterial survival in HD11 macrophages. We compared the transcriptome of ybjX mutations with those of the wild strain and studied the effects of ybjX on miRNA expression in the spleen. Our findings revealed that the mutant strain, ΔybjX, had a lower resistance to chicken serum and blood, as well as lower bacterial survival in HD11 macrophages than AE17. RNA sequencing analyses showed that the ybjX mutation reduced stress resistance by down-regulating mRNAs in metabolic pathways. Infection with the ybjX mutant strain caused changes in the splenic miRNA profile. We verified Kelch repeat and BTB domain-containing protein 11 to be the target of miR-133b. Together, these findings suggest that the ybjX mutation reduces serum, blood, and environmental stress resistance by down-regulating the mRNA in metabolic pathways.

Characterization of the active fragments of Spodoptera litura Lebocin-1.

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.

The roles of kinetochore of micronucleus in mitosis of HeLa cells: a live cell imaging study.

BACKGROUND: Micronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability. However, the roles of kinetochore of MN in mitosis have not been completely addressed. METHODS: The HeLa CENP B-GFP H2B-mCherry cells are applied to address these questions via the long-term live-cell imaging. In the cells, the kinetochore-positive micronucleus (K+MN) contained CENP B-GFP, while the kinetochore-negative micronucleus (K-MN) did not. RESULTS: K-MN-bearing cells produced much more chromosome fragments than did MN-free cells. Most of the chromosome fragments eventually merged into K-MNi. K+MN-bearing cells yielded more kinetochore-positive lagging chromosomes (K+LCs) and K+MNi than MN-free cells did. The results suggested the differences in the fates of K+MNi and K-MNi in mitosis. The cycle of K-MN → Chromosome fragment → K-MN may occur in generations of K-MN-bearing cells, while part of K+MNi might reincorporate into the main nucleus. The K+MN-bearing cells prolonged significantly duration of mitosis compared with MN-free cells. The presence of micronuclei, regardless of K-MN and K+MN, enhanced apoptosis cell death. And K+MN-bearing cells were inclined to apoptosis more than K-MN-bearing cells. The results suggested differences in fates between K-MN-bearing and K+MN-bearing cells. CONCLUSIONS: Kinetochore determined the fates of micronuclei. Kinetochore in micronuclei indirectly prolonged the duration of mitosis. Kinetochore enhanced cytotoxicity of micronuclei. Our data are direct evidences showing the roles of kinetochore of micronucleus in mitosis of HeLa cells.

Curcumin-Mediated Induction of Apoptosis in Human Glioma CHME Cells.

BACKGROUND Curcumin has clear anti-tumor activity in various carcinomas. It regulates various signaling pathways like Wnt/ß-catenin and JAK2/STAT3, which play vital roles in cell proliferation of several carcinomas, but to the best of our knowledge, there are currently no published reports on human glioma CHME cells. Therefore, the aim of this study was to explore the effect of curcumin on human glioma CHME cells. MATERIAL AND METHODS The CHME cell line was purchased from American Type Culture Collection (ATCC). The expressions of caspases 3, caspases 9, PARP, BAX, and BCL2 were detected by Western blot. Annexin V FITC, mitochondrial membrane potential, and reactive oxygen species were detected by flow cytometry. DAPI staining was detected by fluorescence microscopy. Cell viability was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. RESULTS We found that curcumin has cytotoxic activity in human glioma CHME cells, as shown by DAPI staining, annexin V/PI, and nuclear morphology. We found that cell growth decreased with increased concentration of curcumin, as well as sowing effects on expression of caspase-3, caspase-9, and cleavage of PARP, which suggests apoptotic cascade activity. The increase in reactive oxygen species and loss of mitochondrial membrane potential (Δψmt) in concentration-dependent manners suggests biochemical induction of apoptosis in CHME cells. CONCLUSIONS Curcumin has effective anticancer activity in human glioma CHME cells by inducing the apoptotic pathway.

Antimicrobial activities of a proline-rich proprotein from Spodoptera litura.

Antimicrobial peptides (AMPs) are produced by the stimulated humoral immune system. Most mature AMPs contain less than 50 amino acid residues. Some of them are generated from proproteins upon microbial challenges. Here, we report the antimicrobial activities of a proline-rich proprotein, named SlLebocin1 (SlLeb1), from the tobacco cutworm Spodoptera litura. SlLebocin1 cDNA contains a 477-bp open reading frame (ORF). It is mainly expressed in hemocytes and the midgut in naïve larvae. The transcript level was significantly induced in hemocytes but repressed in the midgut and fat body by bacterial challenges. The proprotein contains 158 amino acids with 3 RXXR motifs that are characteristic of some Lepidopteral lebocin proproteins. Four peptides corresponding to the predicted processed fragments were synthesized chemically, and their antimicrobial activities against two Gram-negative and two Gram-positive bacterial strains were analyzed. The peptides showed differential antimicrobial activities. For Escherichia coli and Bacillus subtilis, only the C-terminal fragment (124-158) showed strong inhibitory effects. For Staphylococcus aureus, all peptides showed partial inhibitions. None of them inhibited Serratia marcescens. Bacterial morphologies were examined by the scanning electron microscopy and confocal laser scanning microscopy. The antimicrobial peptides either disrupted cellular membrane or inhibited cell division and caused elongated/enlarged morphologies. The results may provide ideas for designing novel antibiotics.

Effect of gonadotropin-releasing hormone vaccination on T lymphocyte changes in male rats.

The aim of this study was to detect the effect of immunization against gonadotropin-releasing hormone (GnRH) on cell-meditated immunity. Three-week-old male Sprague-Dawley rats (n=32) were randomly and equally assigned to two groups: 1) GnRH-tandem-ovalbumin immunized group; and 2) the control group (injected with an equivalent Al(OH)3 adjuvant). Blood samples were collected at two-week intervals to assess the level of GnRH-specific antibodies and testosterone. Moreover, blood and thymus samples were also collected to analyze the T lymphocyte subpopulations one and two months after the last booster immunization. T lymphocyte immunity against GnRH was activated during the first month post-immunization as exhibited by increased numbers of CD3+ (P<0.05) and CD4+ (P<0.05)T lymphocytes following testosterone suppression (P<0.01), which was then restored and maintained at appropriate levels in the second month. In contrast, the differentiation of T lymphocytes in the thymus was reduced during the first month after immunization as exhibited by the significant decreased number of CD3+ (P<0.05) cells, followed by the restoration and heightened numbers at later time points for both the number of CD3+ (P<0.05) and CD4+ (P<0.01)T lymphocytes. These results suggest that immunization against GnRH interferes with the number of lymphocytes during the early time points following immunization. The number of T lymphocytes initially decreased in the peripheral blood following immunization, but was replenished by newly exported cells from the thymus which eventually restored the T lymphocytes to normal levels.

Effects of GnRH immunization on the reproductive axis and thymulin.

The bidirectional regulation of thymulin in the reproductive-endocrine function of the hypothalamic-pituitary-gonadal (HPG) axis of rats immunized against GnRH remains largely unclear. We explored the alterations in hormones in the HPG axis in immunized rats to dissect the repressive effect of immunization on thymulin, and to clarify the interrelation of reproductive hormones and thymulin in vivo. The results showed that, in the first 2 weeks of booster immunization, thymulin was repressed when reproductive hormones were severely reduced. The self-feedback regulation of thymulin was then stimulated in later immune stages: the rising circulating thymulin upregulated LH and FSH, including GnRH in the hypothalamus, although the levels of those hormones were still significantly lower than in the control groups. In astrocytes, thymulin produced a feedback effect in regulated GnRH neurons. However, in the arcuate nucleus (Arc) and the median eminence (ME), the mediator of astrocytes and other glial cells were also directly affected by reproductive hormones. Thus, in immunized rats, the expression of glial fibrillary acidic protein was distinctly stimulated in the Arc and ME. This study demonstrated that thymulin was downregulated by immunization against GnRH in early stage. Subsequently, the self-feedback regulation was provoked by low circulating thymulin. Thereafter, rising thymulin levels promoted pituitary gonadotropins levels, while acting directly on GnRH neurons, which was mediated by astrocytes in a region-dependent manner in the hypothalamus.

A novel protein elicitor (SsCut) from Sclerotinia sclerotiorum induces multiple defense responses in plants.

In this study, we report the cloning of the SsCut gene encoding cutinase from Sclerotinia sclerotiorum. We isolated a 609-bp cDNA encoding a polypeptide of 202 amino acids with a molecular weight of 20.4 kDa. Heterologous expression of SsCut in Escherichia coli (His-SsCut) caused the formation of lesions in tobacco that closely resembled hypersensitive response lesions. Mutational analysis identified the C-terminal-half peptide and the same amino acids indispensable for both enzyme and elicitor activity. His-SsCut was caused cell death in Arabidopsis, soybean (Glycine max), oilseed rape (Brassica napus), rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum), indicating that both dicot and monocot species are responsive to the elicitor. Furthermore, the elicitation of tobacco was effective in the induction of the activities of hydrogen peroxide, phenylalanine ammonia-lyase, peroxides, and polyphenol oxidase. His-SsCut-treated plants exhibited enhanced resistance as indicated by a significant reduction in the number and size of S. sclerotiorum, Phytophthora sojae, and P. nicotianae lesions on leaves relative to controls. Real-time PCR results indicated that the expression of defense-related genes and genes involved in signal transduction were induced by His-SsCut. Our results demonstrate that SsCut is an elicitor that triggers defense responses in plants and will help to clarify its relationship to downstream signaling pathways that induce defense responses.

Structural and stereochemical studies of alpha-methylene-gamma-lactone-bearing cembrane diterpenoids from a south china sea soft coral Lobophytum crassum.

Four new alpha-methylene-gamma-lactone-bearing cembranoids, 20-acetylsinularolide B (6), presinularolide B (7), 3-dehydroxylpresinularolide B (8), and 3-dehydroxyl-20-acetylpresinularolide B (9), together with five known analogues, sinularolides B-E (1- 4) and 20-acetylsinularolide C (5), were isolated from a South China Sea soft coral Lobophytum crassum. Their structures and relative stereochemistry were established by a combination of detailed spectroscopic data analysis and chemical correlations. The structures of 1- 9 were further confirmed by an X-ray diffraction study on a single crystal of sinularolide B (1). The absolute configurations of sinularolide B (1) and presinularolide B (7) were determined by a novel solid-state CD/TDDFT approach and by a modified Mosher's method, respectively. This study also revealed that the coupling constant between the lactonic methine protons ((3) J 1,2) varies considerably with different functional groups on the cembrane ring and that the determination of the stereochemistry of lactone ring fusion based on this coupling constant is risky. In a bioassay, sinularolides B and C (1 and 2) and new cembranoids 7 and 8 showed in vitro cytotoxicity against the tumor cell lines A-549 and P-388.