Diversity of the Rysto gene conferring resistance to potato virus Y in wild relatives of potato.

BACKGROUND: Potato virus Y (PVY) is among the economically most damaging viral pathogen in production of potato (Solanum tuberosum) worldwide. The gene Rysto derived from the wild potato relative Solanum stoloniferum confers extreme resistance to PVY. RESULTS: The presence and diversity of Rysto were investigated in wild relatives of potato (298 genotypes representing 29 accessions of 26 tuber-bearing Solanum species) using PacBio amplicon sequencing. A total of 55 unique Rysto-like sequences were identified in 72 genotypes representing 12 accessions of 10 Solanum species and six resistant controls (potato cultivars Alicja, Bzura, Hinga, Nimfy, White Lady and breeding line PW363). The 55 Rysto-like sequences showed 89.87 to 99.98% nucleotide identity to the Rysto reference gene, and these encoded in total 45 unique protein sequences. While Rysto-like26 identified in Alicja, Bzura, White Lady and Rysto-like16 in PW363 encode a protein identical to the Rysto reference, the remaining 44 predicted Rysto-like proteins were 65.93 to 99.92% identical to the reference. Higher levels of diversity of the Rysto-like sequences were found in the wild relatives of potato than in the resistant control cultivars. The TIR and NB-ARC domains were the most conserved within the Rysto-like proteins, while the LRR and C-JID domains were more variable. Several Solanum species, including S. antipoviczii and S. hougasii, showed resistance to PVY. This study demonstrated Hyoscyamus niger, a Solanaceae species distantly related to Solanum, as a host of PVY. CONCLUSIONS: The new Rysto-like variants and the identified PVY resistant potato genotypes are potential resistance sources against PVY in potato breeding. Identification of H. niger as a host for PVY is important for cultivation of this plant, studies on the PVY management, its ecology, and migrations. The amplicon sequencing based on PacBio SMRT and the following data analysis pipeline described in our work may be applied to obtain the nucleotide sequences and analyze any full-length genes from any, even polyploid, organisms.

Phosphorylated Hsp27 promotes adriamycin resistance in breast cancer cells through regulating dual phosphorylation of c-Myc.

Chemotherapy resistance of breast cancer cells is one of the major factors affecting patient survival rate. Heat shock protein 27 (Hsp27) is a member of the small heat shock protein family that has been reported to be associated with chemotherapy resistance in tumor cells, but the exact mechanism is not fully understood. Here, we explored the regulation of Hsp27 in adriamycin-resistant pathological conditions of breast cancer in vitro and in vivo. We found that overexpression of Hsp27 in MCF-7 breast cancer cells reversed DNA damage induced by adriamycin, and thereby reduced subsequent cell apoptosis. Non-phosphorylated Hsp27 accelerated ubiquitin-mediated degradation of c-Myc under normal physiological conditions. After stimulation with adriamycin, Hsp27 was phosphorylated and translocated from the cytoplasm into the nucleus, where phosphorylated Hsp27 upregulated c-Myc and Nijmegen breakage syndrome 1 (NBS1) protein levels thus leading to ATM activation. We further showed that phosphorylated Hsp27 promoted c-Myc nuclear import and stabilization by regulating T58/S62 phosphorylation of c-Myc through a protein phosphatase 2A (PP2A)-dependent mechanism. Collectively, the data presented in this study demonstrate that Hsp27, in its phosphorylation state, plays a critical role in adriamycin-resistant pathological conditions of breast cancer cells.

γ-glutamylcysteine alleviates insulin resistance and hepatic steatosis by regulating adenylate cyclase and IGF-1R/IRS1/PI3K/Akt signaling pathways.

Type 2 diabetes mellitus (T2DM), a complex metabolism disease, which was characterized by metabolic disorders including hyperglycemia, has become a major health problem due to the increasing prevalence worldwide. γ-glutamylcysteine (γ-GC) as an immediate precursor of glutathione (GSH) was originally used for the treatment of sepsis, inflammation bowel disease, and senescence. Here, we evaluated the capacity of γ-GC on diabetes-related metabolic parameters in db/db mice and insulin resistance (IR) amelioration in cells induced by palmitic acid (PA). Our data suggested that γ-GC treatment decreased body weight, reduced adipose tissue size, ameliorated ectopic fat deposition in liver, increased the GSH content in liver, improved glucose control and other diabetes-related metabolic parameters in vivo. Moreover, in vitro experiments showed that γ-GC could maintain the balance of free fatty acids (FFAs) and glucose uptake through regulating the translocation of CD36 and GLUT4 from cytoplasm to plasma membrane. Furthermore, our finding also provided evidence that γ-GC could activate Akt not only via adenylate cyclase (AC)/cAMP/PI3K signaling pathway, but also via IGF-1R/IRS1/PI3K signaling pathway to improve IR and hepatic steatosis. Blocking either of two signaling pathways could not activate Akt activation induced by γ-GC. This unique characteristic ensures the important role of γ-GC in glucose metabolism. Collectively, these results suggested that γ-GC could serve as a candidate dipeptide for the treatment of T2DM and related chronic diabetic complications via activating AC and IGF-1R/IRS1/PI3K/Akt signaling pathways to regulate CD36 and GLUT4 trafficking.

Evaluation of reference genes for miRNA and mRNA normalization in tobacco infected with PVYNTN, PVYN-Wi and PVYZ-NTN strains.

This is the first report on identification of the most suitable reference genes for RT-qPCR quantification of miRNA and mRNA in tobacco response to the prevalent recombinant potato virus Y (PVY) strains PVYNTN, PVYN-Wi and the newly identified PVYZ-NTN. Of 10 tested genes, the expression levels of neIF5C, nU2af and nPP2A were the most stable in samples taken from non-inoculated, mock-inoculated, and infected plants at three days post-inoculation (dpi) and 14 dpi. While the homologues of eIF5 were most stably expressed in tobacco in this study and in potato in our previous study (Yin et al., 2021) following inoculation with the same three PVY strains, the homologues of other two genes PP2A and U2af were stably expressed only in tobacco but unstable in potato. The tobacco homologue of PP2A, which was the most stably expressed one in tobacco interaction with PVYNTN, PVYN-Wi and PVYZ-NTN strains in this study, was the least stable one in tobacco interaction with the non-recombinant PVYO strain in a previous study (Baek et al., 2017). This study provides evidence on the influence of host species on expression of housekeeping genes and points out virus strain as a new factor influencing expression stability of reference gene. Caution should be taken when choosing reference genes in gene expression study in Solanaceae hosts response to different PVY strains.

γ-Glutamylcysteine rescues mice from TNBS-driven inflammatory bowel disease through regulating macrophages polarization.

OBJECTIVE: To explore the molecular mechanism of γ-glutamylcysteine (γ-GC) in response to inflammation in vivo and in vitro on regulating the polarization of macrophages. METHODS: The expressions of gene or protein were assessed by qPCR and Western blot assays, respectively. Cell viability was investigated by CCK-8 assay. Eight-week-old male BALB/c mice were established to examine the therapeutic effects of γ-GC in vivo. The release of TNF-α and IL-4 was determined by ELISA assay. Macrophages polarization was identified by flow cytometry assay. RESULTS: Our data showed that γ-GC treatment significantly improved the survival, weight loss, and colon tissue damage of IBD mice. Furthermore, we established M1- and M2-polarized macrophages, respectively, and our findings provided evidence that γ-GC switched M1/M2-polarized macrophages through activating AMPK/SIRT1 axis and inhibiting inflammation-related signaling pathway. CONCLUSION: Collectively, both in vivo and in vitro experiments suggested that γ-GC has the potential to become a promising novel therapeutic dipeptide for the treatment of IBD, which provide new ideas for the treatment of inflammatory diseases in the future.

γ-Glutamylcysteine Exerts Neuroprotection Effects against Cerebral Ischemia/Reperfusion Injury through Inhibiting Lipid Peroxidation and Ferroptosis.

Ferroptosis is a non-apoptotic form of cell death driven by iron-dependent lipid peroxidation. Recent evidence indicates that inhibiting ferroptosis could alleviate cerebral ischemia/reperfusion (CIR) injury. γ-glutamylcysteine (γ-GC), an intermediate of glutathione (GSH) synthesis, can upregulate GSH in brains. GSH is the co-factor of glutathione peroxidase 4 (GPX4), which is the negative regulator of ferroptosis. In this study, we explored the effect of γ-GC on CIR-induced neuronal ferroptosis and brain injury. We found that γ-GC significantly reduced the volume of cerebral infarction, decreased the loss of neurons and alleviated neurological dysfunction induced by CIR in rats. Further observation showed that γ-GC inhibited the CIR-caused rupture of the neuronal mitochondrial outer membrane and the disappearance of cristae, and decreased Fe2+ deposition and lipid peroxidation in rat cerebral cortices. Meanwhile, γ-GC altered the expression of some ferroptosis-related proteins in rat brains. Mechanistically, γ-GC increased the expression of GSH synthetase (GSS) for GSH synthesis via protein kinase C (PKC)ε-mediated activation of nuclear factor erythroid 2-related factor (Nrf2). Our findings suggest that γ-GC not only serves as a raw material but also increases the GSS expression for GSH synthesis against CIR-induced lipid peroxidation and ferroptosis. Our study strongly suggests that γ-GC has potential for treating CIR injury.

γ-Glutamylcysteine ameliorates D-gal-induced senescence in PC12 cells and mice via activating AMPK and SIRT1.

Aging is a natural process accompanied by inflammation and oxidative stress and is closely associated with age-related diseases. As a direct precursor of glutathione, γ-glutamylcysteine (γ-GC) possesses antioxidant and anti-inflammatory properties; however, whether γ-GC plays an important role in anti-aging remains unknown. Here, we investigated the protective effects and mechanisms of γ-GC in D-galactose (D-gal)-induced senescence in PC12 cells and aging mice. Our results showed that γ-GC treatment significantly reduced the percentage of senescence-associated-ß-galactosidase (SA-ß-Gal)-positive cells and inhibited D-gal-induced cell cycle arrest in PC12 cells. The results of Nissl and hematoxylin and eosin (H&E) staining in mouse brain showed that γ-GC treatment markedly reversed the damage in the hippocampus of D-gal-induced aging mice. Moreover, γ-GC increased the phosphorylation of AMP-activated protein kinase (AMPK) to promote the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) while inhibiting the nuclear translocation of deleted in breast cancer 1 (DBC1), which leads to the activation of sirtuin 1 (SIRT1) and deacetylation of p53 in the nucleus. Therefore, γ-GC may be a potential therapeutic candidate compound for the prevention and treatment of age-related diseases.

γ-Glutamylcysteine alleviates ethanol-induced hepatotoxicity via suppressing oxidative stress, apoptosis, and inflammation.

Alcohol abuse is a major cause of alcoholic liver disease (ALD) and can result in fibrosis and cirrhosis. γ-glutamylcysteine (γ-GC) is a precursor of glutathione (GSH) with antioxidant and anti-inflammatory properties. Our research aimed to explore the protective impact of γ-GC on ALD and its potential mechanisms of efficiency in vitro and in vivo. L02 cells were pretreated with γ-GC (20, 40, and 80 µM) for 2 h and exposed to ethanol for 24 h. Cell viability, apoptosis, oxidative stress, and inflammatory levels were measured. The expression of protein cleaved caspase-3 and cleaved PARP and flow cytometry results indicated that γ-GC decreases apoptosis on L02 cells after ethanol treatment. Moreover, γ-GC also attenuated oxidative stress and mitochondrial damage in hepatocytes caused by ethanol via increasing cellular GSH, SOD activity, and mitochondrial membrane potential. In vivo experiments, γ-GC effectively reduced the AST, ALT, and TG levels in mice. The inflammation of ALD was alleviated by γ-GC both in vivo and in vitro. Additionally, histopathological examination demonstrated that γ-GC treatment lessened lipid droplet formation and inflammatory damage. In conclusion, these results showed that γ-GC has anti-inflammatory and anti-apoptotic effects on ALD because it could help hepatocytes maintain sufficient GSH levels to combat the excess reactive oxygen species (ROS) generated during ethanol metabolism. PRACTICAL APPLICATIONS: Alcohol intake is the fifth highest risk factor for premature death and disability among all risk variables. However, few medicines are both safe and effective for the treatment of ALD. As a direct precursor of GSH, γ-GC has a broad variety of potential antioxidant and anti-inflammatory applications for the treatment of numerous medical conditions. In conclusion, these results showed that γ-GC could protect cells from ALD via suppressing oxidative stress, alleviating inflammation, and preventing apoptosis.

A novel mechanism for macrophage pyroptosis in rheumatoid arthritis induced by Pol ß deficiency.

Rheumatoid arthritis (RA) is a chronic and inflammatory autoimmune disease. Macrophage pyroptosis, a proinflammatory form of cell death, is critically important in RA; however, the detailed mechanism underlying pyroptosis induction is not yet well understood. Here, we report that DNA polymerase ß (Pol ß), a key enzyme in base excision repair, plays a pivotal role in RA pathogenesis. Our data shows that Pol ß expression is significantly decreased in peripheral blood mononuclear cells (PBMCs) from active RA patients and collagen-induced arthritis (CIA) mice, and Pol ß deficiency increases the incidence of RA, macrophage infiltration, and bone destruction in CIA mouse models. In vitro, experiments showed that Pol ß deficiency exacerbated macrophage pyroptosis induced by LPS plus ATP, while overexpression of Pol ß inhibited macrophage pyroptosis. Further characterization revealed that Pol ß knockout resulted in DNA damage accumulation and cytosolic dsDNA leakage, which activated the cGAS-STING-NF-κB signaling pathway and upregulated the expression of NLRP3, IL-1 ß, and IL-18. In conclusion, our findings clarify the influence of Pol ß on the development of RA and provide a detailed explanation for the STING-NF-κB pathway to induce macrophage pyroptosis.

γ-Glutamylcysteine attenuates amyloid-ß oligomers-induced neuroinflammation in microglia via blocking NF-κB signaling pathway.

Alzheimer's disease (AD) is the most prevalent neurogenerative disease, characterized by progressive memory loss and cognitive deficits. Intracellular neurofibrillary tangles (NFTs) and amyloid-ß (Aß)-formed neuritic plaques are major pathological features of AD. Aß evokes activation of microglia to release inflammatory mediators and ROS to induce neurotoxicity, leading to neurodegeneration. γ-Glutamylcysteine (γ-GC), an intermediate dipeptide of the GSH-synthesis pathway with anti-inflammatory and anti-oxidative properties, represents a relatively unexplored option for AD treatment. In the present study, we investigated the anti-inflammatory effect of γ-GC on Aß oligomer (AßO)-induced neuroinflammation and the associated molecular mechanism in microglia. The results showed that γ-GC reduced AßO-induced release of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and nitric oxide (NO), and the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). γ-GC decreased ROS and MDA production and increased the GSH level, GSH/GSSG ratio, and SOD activity in AßO-treated microglia. Mechanistically, γ-GC inhibited activation of nuclear factor kappa B (NF-κB), and upregulated the nuclear receptor-related 1 (Nurr1) protein expression to suppress the transcriptional effect of NF-κB on the inflammatory genes. Besides, γ-GC suppressed the AßO-induced neuroinflammation in mice. These findings suggested that γ-GC might represent a potential therapeutic agent for anti-neuroinflammation.

Late blight resistance genes in potato breeding.

MAIN CONCLUSION: Using late blight resistance genes targeting conservative effectors of Phytophthora infestans and the constructing gene pyramids may lead to durable, broad-spectrum resistance, which could be accelerated through genetic engineering. Potato (Solanum tuberosum L.) is one of the most important food crops worldwide. In 2020, potato production was estimated to be more than 359 million tons according to the Food and Agriculture Organization (FAO). Potato is affected by many pathogens, among which Phytophthora infestans, causing late blight, is of the most economic importance. Crop protection against late blight requires intensive use of fungicides, which has an impact on the environment and humans. Therefore, new potato cultivars have been bred using resistance genes against P. infestans (Rpi genes) that originate from wild relatives of potato. Such programmes were initiated 100 years ago, but the process is complex and long. The development of genetic engineering techniques has enabled the direct transfer of resistance genes from potato wild species to cultivars and easier pyramiding of multiple Rpi genes, which potentially increases the durability and spectrum of potato resistance to rapidly evolving P. infestans strains. In this review, we summarize the current knowledge concerning Rpi genes. We also discuss the use of Rpi genes in breeding as well as their detection in existing potato cultivars. Last, we review new sources of Rpi genes and new methods used to identify them and discuss interactions between P. infestans and host.

L-theanine prevents progression of nonalcoholic hepatic steatosis by regulating hepatocyte lipid metabolic pathways via the CaMKKß-AMPK signaling pathway.

BACKGROUND: L-theanine, a non-protein amino acid was found principally in the green tea, has been previously shown to exhibit potent anti-obesity property and hepatoprotective effect. Herein, we investigated the effects of L-theanine on alleviating nonalcoholic hepatic steatosis in vitro and in vivo, and explored the underlying molecular mechanism. METHODS: In vitro, HepG2 and AML12 cells were treated with 500 µM oleic acid (OA) or treated with OA accompanied by L-theanine. In vivo, C57BL/6J mice were fed with normal control diet (NCD), high-fat diet (HFD), or HFD along with L-theanine for 16 weeks. The levels of triglycerides (TG), accumulation of lipid droplets and the expression of genes related to hepatocyte lipid metabolic pathways were detected in vitro and in vivo. RESULTS: Our data indicated that, in vivo, L-theanine significantly reduced body weight, hepatic steatosis, serum levels of alanine transaminase (ALT), aspartate transaminase (AST), TG and LDL cholesterol (LDL-C) in HFD-induced nonalcoholic fatty liver disease (NAFLD) mice. In vitro, L-theanine also significantly alleviated OA induced hepatocytes steatosis. Mechanic studies showed that L-theanine significantly inhibited the nucleus translocation of sterol regulatory element binding protein 1c (SREBP-1c) through AMPK-mTOR signaling pathway, thereby contributing to the reduction of fatty acid synthesis. We also identified that L-theanine enhanced fatty acid ß-oxidation by increasing the expression of peroxisome proliferator-activated receptor α (PPARα) and carnitine palmitoyltransferase-1 A (CPT1A) through AMP-activated protein kinase (AMPK). Furthermore, our study indicated that L-theanine can active AMPK through its upstream kinase Calmodulin-dependent protein kinase kinase-ß (CaMKKß). CONCLUSIONS: Taken together, our findings suggested that L-theanine alleviates nonalcoholic hepatic steatosis by regulating hepatocyte lipid metabolic pathways via the CaMKKß-AMPK signaling pathway.

Protective effect of γ-glutamylcysteine against UVB radiation in NIH-3T3 cells.

BACKGROUND: Ultraviolet (UV) radiation-induced oxidative stress is the main cause of photodamage to the skin. Glutathione (GSH) serves important physiological functions, including scavenging oxygen-free radicals and maintaining intracellular redox balance. γ-glutamylcysteine (γ-GC), as an immediate precursor of GSH and harboring antioxidant and anti-inflammatory properties, represents an unexplored option for skin photodamage treatment. PURPOSE: The purpose of this study was to investigate whether γ-GC can reduce UVB-induced NIH-3T3 cell damage. METHODS: The experimental groups were as follows: control, UVB radiation, UVB radiation after pretreatment with γ-GC. Cell counting kit-8 (CCK-8) assays were used to measure cell proliferation, flow cytometry, and immunoblotting to detect the apoptosis rate and apoptosis-associated proteins. The levels of Reactive Oxygen Species (ROS), Superoxide Dismutase (SOD), and GSH/GSSG (oxidized GSH) were measured to assess oxidative stress. Immunoblotting and immunofluorescence were used to detect DNA damage. The members of the MAPK signaling pathways were detected by immunoblotting. RESULTS: UVB irradiation significantly reduced cell viability and destroyed the oxidative defense system. Pretreatment with γ-GC reduced UVB-induced cytotoxicity, restored the oxidation defense system, and inhibited activation of the MAPK pathway. It also reduced the apoptosis rate, downregulated the levels of cleaved caspase 3 and cleaved PARP. Furthermore, pretreatment with γ-GC reduced the accumulation of γH2AX after UVB radiation exposure, indicating that γ-GC could protect cells from DNA damage. CONCLUSION: γ-GC protected NIH-3T3 from damage caused by UVB irradiation. The photoprotective effect of γ-GC is mediated via strengthening the endogenous antioxidant defense system, which prevents DNA damage and inhibits the activation of the MAPK pathway.

L-Theanine inhibits melanoma cell growth and migration via regulating expression of the clock gene BMAL1.

PURPOSE: L-Theanine is a unique non-protein amino acid found in green tea, which has been identified as a safe dietary supplement. It has been reported that L-theanine exerts various biological activities. In this study, we explored the anti-cancer effects of L-theanine on melanoma cells. METHODS: A375, B16-F10, and PIG1 cell lines were used in the present study. EdU labeling, TUNEL and Annexin V/PI staining, wound-healing, and transwell migration assay were performed to detect the effects of L-theanine on melanoma cell proliferation, apoptosis, and migration. Brain and muscle Arnt-like protein 1 (BMAL1) was knocked down in melanoma cells to evaluate if L-theanine plays the anti-cancer role through regulating circadian rhythm of melanoma cells. The western blot, qRT-PCR, and dual luciferase assay were performed to explore the mechanism involved in the effects of L-theanine on melanoma cells. RESULTS: L-Theanine apparently reduced the viability of melanoma cells. Further experiments showed that L-theanine attenuated the proliferation and migration, and promoted apoptosis of melanoma cells. L-Theanine significantly enhanced the expression of BMAL1, a clock gene in melanoma cells. Down-regulation of BMAL1 suppressed the anti-cancer effects of L-theanine on melanoma cells. Further experiments indicated that the p53 transcriptional activity raised by L-theanine was dependent on BMAL1 expression in melanoma cells. CONCLUSION: L-Theanine exerts the anti-cancer effect on melanoma cells through attenuating the proliferation and migration, and promoting apoptosis of them, which is dependent on the regulation of the clock gene Bmal1 in melanoma cells.

γ-glutamylcysteine suppresses cadmium-induced apoptosis in PC12 cells via regulating oxidative stress.

Cadmium (Cd) is a highly toxic environmental pollutant, leading to the occurrence and development of multiple neurological diseases. γ-glutamylcysteine (γ-GC) is a dipeptide formed by the condensation of l-glutamic acid and l-cysteine, which has antioxidant, anti-inflammatory, and chelating properties. The purpose of this study is to investigate the effect of γ-GC on Cd-induced apoptosis in PC12 cells. PC12 cells were pretreated with or without γ-GC (2 mM or 4 mM) for 2 h and exposed to Cd (10 µM) for 12 h, and survival, apoptosis, and oxidative stress of PC12 cells were detected after different treatments. The results showed that γ-GC significantly inhibited cell viability reduction, apoptosis, and depolarization of mitochondrial transmembrane potential in Cd-treated PC12 cells, as indicated by CCK-8 assay, flow cytometry, TUNEL staining, and JC-1 detection. Western blot showed that γ-GC down-regulated the ratio of Bax/Bcl-2 and the protein levels of cytosolic cytopigment c, cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP. Mechanistically, γ-GC suppressed Cd-induced ROS production, MDA accumulation, and GSH depletion, and increased the activity of antioxidant enzymes. Cd-induced activation of MAPK and PI3K/Akt signaling pathways were inhibited by γ-GC treatment, while sustained phosphorylation of JNK, p38, or Akt reversed anti-apoptotic effects of γ-GC. These results suggested that γ-GC inhibited Cd-induced apoptosis in PC12 cells through decreasing oxidative stress and inhibiting the activation of MAPK and PI3K/Akt signaling pathways. γ-GC could be used as a potential protective agent against Cd neurotoxicity.

GSTpi reduces DNA damage and cell death by regulating the ubiquitination and nuclear translocation of NBS1.

Glutathione S-transferase pi (GSTpi) is an important phase II detoxifying enzyme that participates in various physiological processes, such as antioxidant, detoxification, and signal transduction. The high expression level of GSTpi has been reported to be related to drug-resistant and anti-inflammatory and it functioned via its non-catalytic ligandin. However, the previous protection mechanism of GSTpi in DNA damage has not been addressed so far. Nijmegen breakage syndrome 1 (NBS1) is one of the most important sensor proteins to detect damaged DNA. Here, we investigated the interaction between GSTpi and NBS1 in HEK-293 T cells and human breast adenocarcinoma cells during DNA damage. Our results showed that overexpression of GSTpi in cells by transfecting DNA vector decreased the DNA damage level after methyl methanesulfonate (MMS) or adriamycin (ADR) treatment. We found that cytosolic GSTpi could increase NBS1 ubiquitin-mediated degradation in unstimulated cells, which suggested that GSTpi could maintain the basal level of NBS1 during normal conditions. In response to DNA damage, GSTpi can be phosphorylated in Ser184 and inhibit the ubiquitination degradation of NBS1 mediated by Skp2 to recover NBS1 protein level. Phosphorylated GSTpi can further enhance NBS1 nuclear translocation to activate the ATM-Chk2-p53 signaling pathway. Finally, GSTpi blocked the cell cycle in the G2/M phase to allow more time for DNA damage repair. Thus, our finding revealed the novel mechanism of GSTpi via its Ser184 phosphorylation to protect cells from cell death during DNA damage and it enriches the function of GSTpi in drug resistance.

Indoprofen exerts a potent therapeutic effect against sepsis by alleviating high mobility group box 1-mediated inflammatory responses.

Indoprofen is a non-steroidal anti-inflammatory drug, and has provided insights into treatment of spinal muscular atrophies; however, the treatment effect of indoprofen on sepsis and the precise underlying mechanism remain to be elucidated. This study was carried out to examine the inhibitory effect of indoprofen on high mobility group box 1 (HMGB1)-mediated inflammatory responses in vivo and in vitro. Intraperitoneal injection of indoprofen (20 or 40 mg/kg) at 8 h post-sepsis markedly improved the survival of BALB/c mice and ameliorated multiple-organ injury by blocking the inflammatory responses. In addition, indoprofen partially reduced the HMGB1 level in the serum and in the lung, as well as ameliorated pulmonary edema. Mechanistically, indoprofen potently inhibited the release of HMGB1 following stimulation by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C), and suppressed recombinant human HMGB1(rhHMGB1)-induced inflammatory responses. It was also found that indoprofen has both cyclooxygenase 2-dependent and -independent inhibitory effects on the proinflammatory effect of HMGB1 in THP-1 cells. Further, the drug reduced rhHMGB1-induced cell surface levels of toll-like receptor 2, toll-like receptor 4, and receptor of advanced glycation end-products in a concentration-dependent manner. Collectively, these data demonstrated that the anti-inflammatory effect of indoprofen in sepsis was associated with HMGB1-mediated inflammatory responses, thus offering a favorable mechanistic basis to support the therapeutic potential of indoprofen for the treatment of lethal sepsis or other inflammatory diseases.

Citrate activates autophagic death of prostate cancer cells via downregulation CaMKII/AKT/mTOR pathway.

AIM: The aim of this study was to explore the antitumor effect of citrate on prostate cancer and its underlying mechanism. MAIN METHODS: CCK-8 and Colony formation assay were performed to detect the anti-proliferative effect of citrate on prostate cancer. Flow cytometry analysis was conducted to investigate the pro-apoptosis effect of citrate on prostate cancer. Immunofluorescence assay was taken to detect whether citrate induced autophagy in prostate cancer. Western blot and Immunohistochemical assay were performed to explore the underlying mechanism by which citrate activates autophagic death in prostate cancer cells. Xenograft tumorigenicity assay was conducted to explore whether citrate suppressed the growth of xenograft prostate tumors in vivo. KEY FINDINGS: We found citrate could significantly induce apoptosis and autophagy of prostate cancer cells in vitro and in vivo. Furthermore, treatment with autophagy inhibitor (chloroquine) drastically suppresses the apoptosis rate of prostate cancer induced by citrate. Based on the Ca2+-chelating property of citrate, the further study suggested that citrate activates autophagic cell death in prostate cancer cells via downregulation CaMKII/AKT/mTOR pathway. Finally, citrate suppresses the growth of xenograft prostate tumors without remarkable toxicity in mice. SIGNIFICANCE: Our study elucidated a novel molecular mechanism about the anti-cancer activities of citrate. That citrate activates autophagic cell death of prostate cancer via downregulation CaMKII/AKT/mTOR pathway and without remarkable toxicity in mice. This study suggests that citrate might be a promising therapeutic agent for the treatment of prostate cancer.

L-theanine suppresses the metastasis of prostate cancer by downregulating MMP9 and Snail.

Prostate cancer (PCa) is a very prevalent male-specific malignancy; most PCa patients eventually die as a result of metastasis. L-theanine (C7H14N2O3), a nonprotein amino acid derivative from green tea leaves, has been demonstrated to act as an anticarcinogen through proapoptotic and antiproliferative effects. However, the antimetastatic effect of L-theanine in tumor cells and its underlying mechanism are still unclear. Here, we found that L-theanine could suppress invasion, migration, and increase cell-cell adhesion of prostate cancer cells in vitro and in vivo. We also found that L-theanine could inhibit the epithelial-mesenchymal transition process in PCa. Our study revealed that L-theanine could downregulate MMP9, N-cadherin, Vimentin, Snail, and upregulate E-cadherin. Furthermore, L-theanine suppressed the transcription of MMP9 and Snail by significantly inhibiting the ERK/NF-κB signaling pathway and the binding activity of p65 to the promoter regions of MMP9 and Snail. All of these findings suggest that L-theanine has therapeutic potential for metastatic PCa and may be considered a promising candidate for antimetastatic therapy of prostate cancer.

Reference gene selection for miRNA and mRNA normalization in potato in response to potato virus Y.

This was the first report on evaluating candidate reference genes for quantifying the expression profiles of both coding (e.g., mRNA) and non-coding (e.g., miRNA) genes in potato response to potato virus Y (PVY) inoculation. The reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) method was employed to quantify the expression profiles of eight selected candidate reference genes; their expression stability was analyzed by four statistical algorithms, i.e., geNorm, BestKeeper, NormFinder and RefFinder. The most stable reference genes were sEF1a, sTUBb and seIF5 with a high stability. The least stable ones were sPP2A, sSUI1 and sGAPDH. The same reference gene allows for normalization of both miRNA and mRNA levels from a single RNA sample using cDNAs synthesized in a single RT reaction, in which a stem-loop primer was used for miRNAs and the oligo (dT) for mRNAs.