The circular RNA circ_GRHPR promotes NSCLC cell proliferation and invasion via interactions with the RNA-binding protein PCBP2.

As the most common malignancy, lung cancer is characterised by high rates of occurrence and mortality. Although circular RNAs (circRNAs) are known to act as important regulators in cancer, their role in lung cancer remains poorly understood. In this study, circ_GRHPR expression was found to be significantly upregulated in the serum of five patients with non-small cell lung cancer (NSCLC), compared to that in healthy controls. It is expressed at high levels in NSCLC cell lines, as revealed by qRT-PCR analysis. Functionally, we demonstrated that circ_GRHPR promotes NSCLC proliferation and invasion in vitro and in vivo by cell proliferation, transwell, cell cycle, and tumour-forming assays. Mechanistically, RNA pull-down and RNA immunoprecipitation assays showed that circ_GRHPR interacts with the RNA-binding protein poly(rC)-binding protein 2 (PCBP2) and regulates its subcellular localisation by forming the circ_GRHPR/PCBP2 complex, localizing PCBP2 mainly in the cytoplasm and reducing the proportion found in the nucleus. Furthermore, we demonstrated that four-and-a-half LIM-only protein 3 (FHL3) is a tumour-stimulating factor in NSCLC that interacts with and is influenced by PCBP2. Circ_GRHPR increased FHL3 expression in the nucleus of NSCLC cells by decreasing PCBP2 expression therein and promoting the proliferation and invasion of NSCLC cells. Therefore, our study identified that circ_GRHPR promotes NSCLC proliferation and invasion, providing a possible explanation for its mechanism of action.

Theophylline inhibits cigarette smoke-induced inflammation in skeletal muscle by upregulating HDAC2 expression and decreasing NF-κB activation.

Inflammation is associated with skeletal muscle dysfunction and atrophy in patients with chronic obstructive pulmonary disease (COPD). Theophylline has an anti-inflammatory role in COPD. However, the effects of theophylline on inflammation in skeletal muscle in COPD have rarely been reported. The aims of this study were to explore whether theophylline has an anti-inflammatory effect on skeletal muscle in a mouse model of emphysema and to investigate the molecular mechanism underlying this effect. In mice, cigarette smoke (CS) exposure for 28 wk resulted in atrophy of the gastrocnemius muscle. Histone deacetylase 2 (HDAC2) and nuclear factor-κBp65 (NF-κBp65) mRNA and protein levels were significantly decreased and increased, respectively, in gastrocnemius muscle. This effect was revered by aminophylline. The exposure of murine skeletal muscle C2C12 cells to CS extract (CSE) significantly increased IL-8 and TNF-α levels as well as NF-κBp65 mRNA and protein levels and NF-κBp65 activity. This effect was reversed by theophylline. HDAC2 knockdown enhanced the activity of NF-κBp65 and increased IL-8 and TNF-α levels in C2C12 cells. CSE significantly increased the interaction of HDAC2 with NF-κBp65 in C2C12 cells. These data suggest that theophylline has an anti-inflammatory effect on skeletal muscle in a mouse model of emphysema by upregulating HDAC2 expression and decreasing NF-κBp65 activation.

Septic pulmonary embolism in China: clinical features and analysis of prognostic factors for mortality in 98 cases.

BACKGROUND: To investigate the clinical features of septic pulmonary embolism (SPE) cases and prognostic factors for in-hospital mortality in China. METHODS: A retrospective analysis was conducted of SPE patients hospitalized between January 2007 and June 2018 in the Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Guangxi Medical University. RESULTS: A total of 98 patients with SPE were identified. All patients had bilateral multiple peripheral nodules on chest computed tomography. The most common pathogen found in blood culture was Staphylococcus aureus (10/33, 30.3%). Transthoracic echocardiography was performed in 39 patients and 20 showed vegetations. Bronchoscopy was performed in 24 patients. Bronchoalveolar lavage fluid (BALF) was obtained from 15 patients (62.5%) and showed predominantly polymorphonuclear cell infiltration (52%, range of 48%~ 63%). Four patients received transbronchial lung biopsy, and histopathological examinations revealed suppurative pneumonia and organizing pneumonia. The in-hospital mortality rate was 19.4%. Age (odds ratio [OR] 1.100; 95% confidence interval [CI] 1.035-1.169), hypotension (OR 7.260; 95% CI 1.126-46.804) and ineffective or delay of empirical antimicrobial therapy (OR 7.341; 95% CI 1.145-47.045) were found to be independent risk factors for in-hospital mortality, whereas drainage treatment was found to be a protective factor (OR 0.33; 95% CI 0.002-0.677). CONCLUSIONS: SPE cases presented with nonspecific clinical manifestations and radiologic features. Blood cultures and bronchoscopy are important measures for early diagnosis and differential diagnosis. There is relationship between primary infection sites and the type of pathogen. Maintaining normal blood pressure and providing timely and appropriate initial antimicrobial therapy for effective control of the infection could improve prognosis.

Acute Penicillium marneffei infection stimulates host M1/M2a macrophages polarization in BALB/C mice.

BACKGROUND: Penicillium marneffei (P. marneffei) is a thermally dimorphic fungus pathogen that causes fatal infection. Alveolar macrophages are innate immune cells that have critical roles in protection against pulmonary fungal pathogens and the macrophage polarization state has the potential to be a deciding factor in disease progression or resolution. The aim of this study was to investigate mouse alveolar macrophage polarization states during P. marneffei infection. RESULTS: We used enzyme-linked immunosorbent (ELISA) assays, quantitative real-time PCR (qRT-PCR), and Griess, arginase activity to evaluate the phenotypic markers of alveolar macrophages from BALB/C mice infected with P. marneffei. We then treated alveolar macrophages from infected mice with P. marneffei cytoplasmic yeast antigen (CYA) and investigated alveolar macrophage phenotypic markers in order to identify macrophage polarization in response to P. marneffei antigens. Our results showed: i) P. marneffei infection significantly enhanced the expression of classically activated macrophage (M1)-phenotypic markers (inducible nitric oxide synthase [iNOS] mRNA, nitric oxide [NO], interleukin-12 [IL-12], tumor necrosis factor-alpha [TNF-α]) and alternatively activated macrophage (M2a)-phenotypic markers (arginase1 [Arg1] mRNA, urea) during the second week post-infection. This significantly decreased during the fourth week post-infection. ii) During P. marneffei infection, CYA stimulation also significantly enhanced the expression of M1 and M2a-phenotypic markers, consistent with the results for P. marneffei infection and CYA stimulation preferentially induced M1 subtype. CONCLUSIONS: The data from the current study demonstrated that alveolar macrophage M1/M2a subtypes were present in host defense against acute P. marneffei infection and that CYA could mimic P. marneffei to induce a host immune response with enhanced M1 subtype. This could be useful for investigating the enhancement of host anti-P. marneffei immune responses and to provide novel ideas for prevention of P. marneffei-infection.

Over-expression of an inactive mutant cathepsin D increases endogenous alpha-synuclein and cathepsin B activity in SH-SY5Y cells.

Parkinson's disease is a neurodegenerative movement disorder. The histopathology of Parkinson's disease comprises proteinaceous inclusions known as Lewy bodies, which contains aggregated α-synuclein. Cathepsin D (CD) is a lysosomal protease previously demonstrated to cleave α-synuclein and decrease its toxicity in both cell lines and mouse brains in vivo. Here, we show that pharmacological inhibition of CD, or introduction of catalytically inactive mutant CD, resulted in decreased CD activity and increased cathepsin B activity, suggesting a possible compensatory response to inhibition of CD activity. However, this increased cathepsin B activity was not sufficient to maintain α-synuclein degradation, as evidenced by the accumulation of endogenous α-synuclein. Interestingly, the levels of LC3, LAMP1, and LAMP2, proteins involved in autophagy-lysosomal activities, as well as total lysosomal mass as assessed by LysoTracker flow cytometry, were unchanged. Neither autophagic flux nor proteasomal activities differs between cells over-expressing wild-type versus mutant CD. These observations point to a critical regulatory role for that endogenous CD activity in dopaminergic cells in α-synuclein homeostasis which cannot be compensated for by increased Cathepsin B. These data support the potential need to enhance CD function in order to attenuate α-synuclein accumulation as a therapeutic strategy against development of synucleinopathy.

Down-regulation of Aquaporin1 (AQP1) by peptidoglycan via p38 MAPK pathways in primary rat pleural mesothelial cells.

BACKGROUND AND OBJECTIVE: This study was designed to investigate the p38 mitogen-activated protein kinase (MAPK) signaling pathway involved in Aquaporin1 (AQP1) expression caused by staphylococcal peptidoglycan (PGN) in cultured rat pleural mesothelial cells (rPMCs) in vitro. METHODS: RT-PCR and immunoblot analysis were used to determine the relative mRNA and protein levels of AQP1 by PGN in rPMCs. P38 kinase inhibitor SB203580, JNK inhibitor SP600125, and ERK1/2 inhibitor PD98059 were used to determine the effects of PGN-induced AQP1 expression by immunoblot. Activation of p38 by PGN was reflected by detecting the phosphorylation constituent of p38, using immunoblot. The shift of localization after activation of p38 by PGN was investigated by immunofluorescence assay. RESULTS: AQP1 transcription and protein expression were decreased by PGN in dose-dependent and time-dependent manners in rPMCs. Down-regulation of AQP1 by PGN was blocked only by SB203580, neither by SP600125 nor by PD98059. Furthermore, rPMCs exposed to PGN showed activation of p38 MAPK. Phospho-p38 protein production was increased by PGN stimulation in rPMCs. The localization of phospho-p38 was both in the cytosol and nuclei after PGN treatment, while its normal distribution is mainly in the cytosol in rPMCs. CONCLUSION: AQP1 expression was decreased by PGN in both dose-dependent and time-dependent manners in rPMCs. This down-regulation by PGN-induced AQP1 in rPMCs may be mediated by the activation of p38 MARK pathway.

Bioenergetic and autophagic control by Sirt3 in response to nutrient deprivation in mouse embryonic fibroblasts.

Sirt3 (sirtuin 3) is an NAD-dependent deacetylase localized to mitochondria. Sirt3 expression is increased in mouse muscle and liver by starvation, which could protect against the starvation-dependent increase in oxidative stress and protein damage. Damaged proteins and organelles depend on autophagy for removal and this is critical for cell survival, but the role of Sirt3 is unclear. To examine this, we used Sirt3-KO (knockout) mouse embryonic fibroblast cells, and found that, under basal conditions, Sirt3-KO cells exhibited increased autophagy flux compared with WT (wild-type) cells. In response to nutrient deprivation, both WT and KO cells exhibited increased basal and ATP-linked mitochondrial respiration, indicating an increased energy demand. Both cells exhibited lower levels of phosphorylated mTOR (mammalian target of rapamycin) and higher autophagy flux, with KO cells exhibiting lower maximal mitochondrial respiration and reserve capacity, and higher levels of autophagy than WT cells. KO cells exhibit higher phospho-JNK (c-Jun N-terminal kinase) and phospho-c-Jun than WT cells under starvation conditions. However, inhibition of JNK activity in Sirt3-KO cells did not affect LC3-I (light chain 3-I) and LC3-II levels, indicating that Sirt3-regulated autophagy is independent of the JNK pathway. Caspase 3 activation and cell death are significantly higher in Sirt3-KO cells compared with WT cells in response to nutrient deprivation. Inhibition of autophagy by chloroquine exacerbated cell death in both WT and Sirt3-KO cells, and by 3-methyadenine exacerbated cell death in Sirt3-KO cells. These data suggest that nutrient deprivation-induced autophagy plays a protective role in cell survival, and Sirt3 decreases the requirement for enhanced autophagy and improves cellular bioenergetics.

Comprehensive Analysis and Experimental Validation of TLL2 as a Potential New Prognostic Biomarker Associated with Immune Infiltration in Lung Adenocarcinoma.

BACKGROUND: The precise function of Tolloid Like 2 (TLL2) remains uncertain within the context of Lung Adenocarcinoma (LUAD). OBJECTIVE: The primary objective of this investigation was to conduct a thorough analysis. METHODS: To assess its diagnostic utility, data from The Cancer Genome Atlas (TCGA) database were used to assess TLL2 expression in pan-cancer and LUAD. The study has also investigated the correlation between TLL2 expression levels and LUAD symptoms and prognosis. Furthermore, the study has explored possible regulatory networks involving TLL2, including its association with immune infiltration, tumor stemness index (mRNAsi), and drug sensitivity in LUAD. We have explored TLL2 expression in single-cell sequencing of LUAD and the genomic variation and clinical significance of TLL2 in LUAD. The expression of TLL2 has been validated in GSE87340 and cell lines by quantitative Real-time PCR (qRT-PCR). RESULTS: An abnormal expression of TLL2 has been found in pan-cancer and LUAD. In LUAD patients, elevated levels of TLL2 were significantly related to the T stage (p = 0.046) and the pathological stage (p = 0.016). The expression of TLL2 in patients with LUAD was significantly associated with poorer Overall Survival (OS) (p < 0.001). The expression of TLL2 was determined to be an independent predictor of poorer OS (p = 0.042). TLL2 was associated with ribosome, neuroactive ligand-receptor interaction, allograft rejection, ECM receptor interaction, asthma, porphyrin and chlorophyll metabolism, focal adhesion, pentose and glucuronate inter-conversions, and ascorbate and aldarate metabolism. The expression of TLL2 in LUAD was correlated with immune infiltration and mRNAsi. The expression of TLL2 was significantly and negatively correlated with TAK-715, XMD13-2, STF-62247, OSI-930, and EHT-1864 in LUAD. The TLL2 gene was up- -regulated in multiple individual LUAD cells. LUAD patients with altered TLL2 had a shorter PFS as opposed to those with unaltered TLL2. The expression of TLL2 was significantly increased in LUAD cells. CONCLUSION: For patients with LUAD, TLL2 may serve as an immunotherapeutic target and a useful prognosis biomarker.

Low expression of GOT2 promotes tumor progress and predicts poor prognosis in hepatocellular carcinoma.

Background: To explore the biological function and the underlying mechanisms of GOT2 in hepatocellular carcinoma (HCC). Materials & methods: The expression level and prognostic value of GOT2 were examined using International Cancer Genome Consortium and International Cancer Proteogenome Consortium databases. The cell counting kit-8 method, clone formation, Transwell® assays and western blotting were used to evaluate the effects of GOT2 on the biological function and autophagy of HCC cells. Results: The expression of GOT2 was downregulated in HCC tissues and correlated with poor prognosis of HCC patients. Knockdown of GOT2 promoted proliferation, migration and invasion of HCC cells and promoted cells' proliferation by inducing autophagy. Conclusion: GOT2 plays a tumor-inhibitory role in HCC and may be a potential therapeutic target for HCC.

Reduced CaM/FLIP binding by a single point mutation in c-FLIP(L) modulates Fas-mediated apoptosis and decreases tumorigenesis.

We have previously demonstrated that calmodulin (CaM) binds directly to c-FLIP(L) in a Ca(2+)-dependent manner. Deletion of the CaM-binding region (amino acid 197-213) results in reduced CaM binding, and increased Fas-mediated apoptosis and decreased tumorigenesis of cholangiocarcinoma cells. The present studies were designed to identify the precise amino acids between 197 and 213 that are responsible for CaM/FLIP binding, and their roles in mediating the anti-apoptotic function of c-FLIP(L). Sequence analysis of the CaM-binding region at 197-213 predicted three unique positively charged residues at 204, 207 and 209, which might be responsible for the CaM/FLIP binding. A point mutation at H204 of c-FLIP(L) was found to markedly reduce CaM binding, whereas point mutation at R207 or K209 did not affect c-FLIP(L) binding to CaM. Decreased CaM/FLIP binding was confirmed in cholangiocarcinoma cells overexpressing the H204 c-FLIP(L) mutant. Reduced CaM binding by the H204 mutant resulted in increased sensitivity to Fas-mediated apoptosis and inhibited tumor growth in mice compared with wild-type c-FLIP(L). Death-inducing signaling complex (DISC) analysis showed that the reduced CaM binding to H204 mutant resulted in less c-FLIP(L) recruited into the DISC. Concurrently, increased caspase 8 was recruited to the DISC, which resulted in increased cleavage and activation of caspase 8, activation of downstream caspase 3 and increased apoptosis. Therefore, these results demonstrate that the H204 residue is responsible for c-FLIP(L) binding to CaM, which mediates the anti-apoptotic function of c-FLIP(L), most likely through affecting recruitment of caspase 8 into the DISC and thus caspase 8 activation. These studies further characterized CaM/FLIP interaction and its function in regulating Fas-mediated apoptosis and tumorigenesis, which may provide new therapeutic targets for cancer therapy.

RNA sequencing identifies two novel liver-specific long noncoding RNAs with potential diagnostic value in hepatocellular carcinoma.

Aim: To explore the expression profiles of long noncoding RNAs (lncRNAs) and identify novel lncRNAs as biomarkers for early diagnosis and therapy of hepatocellular carcinoma (HCC). Materials & methods: Expression profiles of lncRNAs and mRNAs in five paired HCC and adjacent normal tissues were obtained by RNA sequencing. Eight lncRNAs, including two novel liver-specific lncRNAs (NONHSAT059247.2 and NONHSAT013897.2), were validated in another 74 pairs of HCC and adjacent normal tissues by quantitative reverse transcription PCR. Results: The results of quantitative reverse transcription PCR showed that NONHSAT252133.1, NONHSAT112116.2 and NONHSAT242657.1 were significantly upregulated in HCC tissues, whereas NONHSAT169790.1, NONHSAT059247.2 and NONHSAT013897.2 were significantly downregulated. Two liver-specific lncRNAs demonstrated excellent diagnostic performance: NONHSAT059247.2 (area under the curve = 0.941, 95% CI: 0.902-0.979, p < 0.0001), NONHSAT013897.2 (area under the curve = 0.944, 95% CI: 0.906-0.983, p < 0.0001). Conclusion: The liver-specific lncRNAs NONHSAT059247.2 and NONHSAT013897.2, may provide new biomarkers for the future study on diagnosis, therapy and mechanisms of HCC.

Predicting cancer-specific mortality in patients with parotid gland carcinoma by competing risk nomogram.

BACKGROUND: The aim of the present study was to establish a competing risk nomogram to predict parotid gland cancer-specific mortality (PGC-SM). METHODS: Seven thousand nine hundred and sixty-two patients extracted from SEER database were randomly categorized into training and validation sets. The competing risk model was used to identify factors associated with PGC-SM. The nomogram was evaluated via concordance indexes (C-indexes), calibration plots, and decision curve analysis (DCA). RESULTS: Male, elderly, white, widowed, larger tumor, no surgery, advanced tumor grade, lymph node (LN) metastasis, adenocarcinoma (ADC), and higher TNM stage were associated with higher incidence of PGC-SM. Calibration plots showed that the nomogram was well calibrated. C-indexes for nomogram were 0.84 (95% CI: 0.81-0.86) and 0.84 (95% CI: 0.82-0.86) in training and validation sets, respectively. DCA demonstrated the clinical usefulness of nomogram. CONCLUSIONS: The competing risk nomogram shows high performance in predicting PGC-SM, which might enable clinicians formulate suitable treatment protocols for patients with parotid gland carcinoma (PGC).

Integrated profiling identifies ITGB3BP as prognostic biomarker for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor. In this study, we sought to identify a novel biomarker for HCC by analyzing transcriptome and clinical data. The R software was used to analyze the differentially expressed genes (DEGs) in the datasets GSE74656 and GSE84598 downloaded from the Gene Expression Omnibus database, followed by a functional annotation. A total of 138 shared DEGs were screened from two datasets. They were mainly enriched in the "Metabolic pathways" pathway (Padj = 8.21E-08) and involved in the carboxylic acid metabolic process (Padj = 0.0004). The top 10 hub genes were found by protein-protein interaction analysis and were upregulated in HCC tissues compared to normal tissues in The Cancer Genome Atlas database. Survival analysis distinguished 8 hub genes CENPE, SPDL1, Hyaluronan-mediated motility receptor, Rac GTPase activating protein 1, Thyroid hormone receptor interactor 13, cytoskeleton-associated protein (CKAP) 2, CKAP5, and Integrin subunit beta 3 binding protein (ITGB3BP) were considered as prognostic hub genes. Multivariate cox regression analysis indicated that all the prognostic hub genes were independent prognostic factors for HCC. Furthermore, the receiver operating characteristic curve revealed that the 8-hub genes model had better prediction performance for overall survival compared to the T stage (p = 0.008) and significantly improved the prediction value of the T stage (p = 0.002). The Human Protein Atlas showed that the protein expression of ITGB3BP was upregulated in HCC, so the expression of ITGB3BP was further verified in our cohort. The results showed that ITGB3BP was upregulated in HCC tissues and was significantly associated with lymph node metastasis.

CCL22 recruits CD4-positive CD25-positive regulatory T cells into malignant pleural effusion.

PURPOSE: The aim of this study was to explore the presence of the chemokines CCL22 and CCL17 in malignant pleural effusion, and the chemoattractant activity of these chemokines on CD4-positive CD25-positive Foxp3-positive regulatory T cells infiltrating into the pleural space. EXPERIMENTAL DESIGN: The concentrations of CCL22 and CCL17 in both pleural effusions and sera from 33 patients with lung cancer were determined. Flow cytometry was done to determine T lymphocyte subsets in cell pellets of pleural effusion. Pleural cells were analyzed for the expression of CCL22 and CCL17. The chemoattractant activity of CCL22 for regulatory T cells in vitro and in vivo was also observed. RESULTS: The concentration of CCL22 in malignant pleural effusion was significantly higher than that in the corresponding serum. Pleural fluid from lung cancer patients was chemotactic for regulatory T cells, and this activity was partly blocked by an anti-CCL22, but not by an anti-CCL17 antibody. Intrapleural administration of CCL22 of patients produced a marked progressive influx of regulatory T cells into pleural space. CONCLUSIONS: Compared with serum, CCL22 seemed to be increased in malignant pleural effusion, and could directly induce regulatory T cell infiltration into the pleural space in patients with malignant effusion.

Copper and manganese induce yeast apoptosis via different pathways.

Metal ions are essential as well as toxic to the cell. The mechanism of metal-induced toxicity is not well established. Here, for the first time we studied two essential nutritional elements, copper and manganese, for their apoptotic effects in yeast Saccharomyces cerevisiae. Although beneficial at subtoxic levels, we demonstrated that at moderately toxic levels, both metals induce extensive apoptosis in yeast cells. At even higher concentrations, necrosis takes over. Furthermore, we investigated the molecular pathways mediating Cu- and Mn-mediated apoptotic action. Mitochondria-defective yeast exhibit a much reduced apoptotic marker expression and better survival under Cu and Mn stress, indicating mitochondria are involved in both Cu- and Mn-induced apoptosis. Reactive oxygen species (ROS) are generated in high amounts in Cu- but not in Mn-induced cell death, and Cu toxicity can be alleviated by overexpression of superoxide dismutase 2, suggesting ROS mediate Cu but not Mn toxicity. Yeast metacaspase Yca1p is not involved in Cu-induced apoptosis, although it plays an important role in the Mn-induced process. A genetic screen identified Cpr3p, a yeast cyclophilin D homologue, as mediating the Cu-induced apoptotic program. Cpr3p mutant seems to eliminate Cu-induced apoptosis without affecting ROS production, while leaving necrosis intact. These results may provide important insight into a detailed understanding at the molecular and cellular level of metal toxicity and metal accumulation diseases.

Big Data-Based Identification of Multi-Gene Prognostic Signatures in Liver Cancer.

Simultaneous identification of multiple single genes and multi-gene prognostic signatures with higher efficacy in liver cancer has rarely been reported. Here, 1,173 genes potentially related to the liver cancer prognosis were mined with Coremine, and the gene expression and survival data in 370 samples for overall survival (OS) and 319 samples for disease-free survival (DFS) were retrieved from The Cancer Genome Atlas. Numerous survival analyses results revealed that 39 genes and 28 genes significantly associated with DFS and OS in liver cancer, including 18 and 12 novel genes that have not been systematically reported in relation to the liver cancer prognosis, respectively. Next, totally 9,139 three-gene combinations (including 816 constructed by 18 novel genes) for predicting DFS and 3,276 three-gene combinations (including 220 constructed by 12 novel genes) for predicting OS were constructed based on the above genes, and the top 15 of these four parts three-gene combinations were selected and shown. Moreover, a huge difference between high and low expression group of these three-gene combination was detected, with median survival difference of DFS up to 65.01 months, and of OS up to 83.57 months. The high or low expression group of these three-gene combinations can predict the longest prognosis of DFS and OS is 71.91 months and 102.66 months, and the shortest is 6.24 months and 13.96 months. Quantitative real-time polymerase chain reaction and immunohistochemistry reconfirmed that three genes F2, GOT2, and TRPV1 contained in one of the above combinations, are significantly dysregulated in liver cancer tissues, low expression of F2, GOT2, and TRPV1 is associated with poor prognosis in liver cancer. Overall, we discovered a few novel single genes and multi-gene combinations biomarkers that are closely related to the long-term prognosis of liver cancer, and they can be potential therapeutic targets for liver cancer.

Caspase-independent apoptosis in yeast.

Apoptosis is a highly regulated cellular suicide program crucial for metazoan development. Yeast counterparts of central metazoan apoptotic regulators, such as metacaspase Yca1p, have been identified. In spite of the importance of Yca1p in yeast apoptotic process, many other factors such as Aif1p, orthologs of EndoG, AMID and cyclophilin D play important roles in caspase-independent apoptotic pathways. This review summarized recent progress about studies of various intrinsic and extrinsic apoptotic stimuli that may induce yeast cell death via caspase-independent apoptosis.

Yeast AMID homologue Ndi1p displays respiration-restricted apoptotic activity and is involved in chronological aging.

Apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) are both mitochondrial flavoproteins that trigger caspase-independent apoptosis. Phylogenetic analysis suggests that these two proteins evolutionarily diverge back from their common prokaryote ancestor. Compared with AIF, the proapoptotic nature of AMID and its mode of action are much less clarified. Here, we show that overexpression of yeast AMID homologue internal NADH dehydrogenase (NDI1), but not external NADH dehydrogenase (NDE1), can cause apoptosis-like cell death, and this effect can be repressed by increased respiration on glucose-limited media. This result indicates that the regulatory network of energy metabolism, in particular the cross-talk between mitochondria and the rest of the cell, is involved in Ndi1p-induced yeast cell apoptosis. The apoptotic effect of NDI1 overexpression is associated with increased production of reactive oxygen species (ROS) in mitochondria. In addition, NDI1 overexpression in sod2 background causes cell lethality in both fermentable and semifermentable media. Interruption of certain components in the electron transport chain can suppress the growth inhibition from Ndi1p overexpression. We finally show that disruption of NDI1 or NDE1 decreases ROS production and elongates the chronological life span of yeast, accompanied by the loss of survival fitness. Implication of these findings for Ndi1p-induced apoptosis is discussed.

SENP2 Suppresses Necdin Expression to Promote Brown Adipocyte Differentiation.

Brown adipose tissue (BAT) is a thermogenic organ that maintains body temperature and energy homeostasis. Transcriptional regulation plays an important role in the program of brown adipogenesis. However, it remains unclear how the transcriptional events are controlled in this program. In this study, we analyze an SENP2 BAT conditional knockout mouse model and find that SENP2-mediated de-SUMOylation is essential for BAT development. SENP2 catalyzes de-SUMOylation of cAMP response element-binding protein (CREB) to suppress Necdin expression, which induces brown adipocyte differentiation and brown adipogenesis. Mechanistically, we find that SUMOylation enhances CREB interaction with serine/threonine protein phosphatase 2A (PP2A) to de-phosphorylate CREB, which activates Necdin transcription. SENP2 deficiency enhances the expression of Necdin to inhibit brown adipocyte differentiation. Therefore, we reveal a crucial role of SENP2-mediated de-SUMOylation of CREB in suppression of Necdin expression during brown adipose development and brown adipogenesis.

Targeted disruption of tyrosinase causes melanin reduction in Carassius auratus cuvieri and its hybrid progeny.

The white crucian carp (Carassius auratus cuvieri, WCC) not only is one of the most economically important fish in Asia, characterized by strong reproductive ability and rapid growth rates, but also represents a good germplasm to produce hybrid progenies with heterosis. Gene knockout technique provides a safe and acceptant way for fish breeding. Achieving gene knockout in WCC and its hybrid progeny will be of great importance for both genetic studies and hybridization breeding. Tyrosinase (TYR) is a key enzyme in melanin synthesis. Depletion of tyr in zebrafish and mice results in mosaic pigmentation or total albinism. Here, we successfully used CRISPR-Cas9 to target tyr in WCC and its hybrid progeny (WR) derived from the cross of WCC (♀) and red crucian carp (Carassius auratus red var., RCC, ♂). The level of TYR protein was significantly reduced in mutant WCC. Both the mutant WCC and the mutant WR showed different degrees of melanin reduction compared with the wild-type sibling control fish, resulting from different mutation efficiency ranging from 60% to 90%. In addition, the transcriptional expression profiles of a series of pivotal pigment synthesis genes, i.e. tyrp1, mitfa, mitfb, dct and sox10, were down-regulated in tyr-CRISPR WCC, which ultimately caused a reduction in melanin synthesis. These results demonstrated that tyr plays a key role in melanin synthesis in WCC and WR, and CRISPR-Cas9 is an effective tool for modifying the genome of economical fish. Furthermore, the tyr-CRISPR models could be valuable in understanding fundamental mechanisms of pigment formation in non-model fish.