Alcohol Abstinence Rescues Hepatic Steatosis and Liver Injury via Improving Metabolic Reprogramming in Chronic Alcohol-Fed Mice.

Background: Alcoholic liver disease (ALD) caused by chronic ethanol overconsumption is a common type of liver disease with a severe mortality burden throughout the world. The pathogenesis of ALD is complex, and no effective clinical treatment for the disease has advanced so far. Prolonged alcohol abstinence is the most effective therapy to attenuate the clinical course of ALD and even reverse liver damage. However, the molecular mechanisms involved in alcohol abstinence-improved recovery from alcoholic fatty liver remain unclear. This study aims to systematically evaluate the beneficial effect of alcohol abstinence on pathological changes in ALD. Methods: Using the Lieber-DeCarli mouse model of ALD, we analysed whether 1-week alcohol withdrawal reversed alcohol-induced detrimental alterations, including oxidative stress, liver injury, lipids metabolism, and hepatic inflammation, by detecting biomarkers and potential targets. Results: Alcohol withdrawal ameliorated alcohol-induced hepatic steatosis by improving liver lipid metabolism reprogramming via upregulating phosphorylated 5'-AMP -activated protein kinase (p-AMPK), peroxisome proliferator-activated receptor-α (PPAR-α), and carnitine palmitoyltransferase-1 (CPT-1), and downregulating fatty acid synthase (FAS) and diacylglycerol acyltransferase-2 (DGAT-2). The activities of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-px), were significantly enhanced by alcohol withdrawal. Importantly, the abstinence recovered alcohol-fed induced liver injury, as evidenced by the improvements in haematoxylin and eosin (H&E) staining, plasma alanine aminotransferase (ALT) levels, and liver weight/body weight ratio. Alcohol-stimulated toll-like receptor 4/mitogen-activated protein kinases (TLR4/MAPKs) were significantly reversed by alcohol withdrawal, which might mechanistically contribute to the amelioration of liver injury. Accordingly, the hepatic inflammatory factor represented by tumour necrosis factor-alpha (TNF-α) was improved by alcohol abstinence. Conclusion: In summary, we reported that alcohol withdrawal effectively restored hepatic lipid metabolism and reversed liver injury and inflammation by improving metabolism reprogramming. These findings enhanced our understanding of the biological mechanisms involved in the beneficial role of alcohol abstinence as an effective treatment for ALD.

Characterization of the complete chloroplast genome of Polygonatum sibiricum (Liliaceae), a well-known herb to China.

Polygonatum sibiricum is a famous and well-known TCH (Traditional Chinese Herb) in China. In this paper, the complete chloroplast genome of P. sibiricum was studied and illustrated to add more genetic information and data. The chloroplast genome is 152,960 bp in length and a typical quadripartite structure, which exhibits a large single-copy region (LSC) of 81,471 bp, a small single-copy region (SSC) of 18,485 bp and a pair of inverted-repeat regions (IRs) of 26,502 bp in each. The overall nucleotide composition of chloroplast genome is: 30.7% A, 31.4% T, 19.3% C, 18.6% G and the total GC content 37.9%. A total of 136 genes were annotated that included 90 protein-coding genes (PCGs), 38 transfer RNA (tRNAs) and 8 ribosome RNA (rRNAs). The phylogenetic ML tree shown that P. sibiricum is closely related to P. cyrtonema on genetic position relationship by the Maximum-Likelihood (ML) method.

Identification and functional characterization of ABCC transporters for Cd tolerance and accumulation in Sedum alfredii Hance.

Cd is one of the potential toxic elements (PTEs) exerting great threats on the environment and living organisms and arising extensive attentions worldwide. Sedum alfredii Hance, a Cd hyperaccumulator, is of great importance in studying the mechanisms of Cd hyperaccumulation and has potentials for phytoremediation. ATP-binding cassette sub-family C (ABCC) belongs to the ABC transporter family, which is deemed to closely associate with multiple physiological processes including cellular homeostasis, metal detoxification, and transport of metabolites. In the present work, ten ABCC proteins were identified in S. alfredii Hance, exhibiting uniform domain structure and divergently clustering with those from Arabidopsis. Tissue-specific expression analysis indicated that some SaABCC genes had significantly higher expression in roots (Sa23221 and Sa88F144), stems (Sa13F200 and Sa14F98) and leaves (Sa13F200). Co-expression network analysis using these five SaABCC genes as hub genes produced two clades harboring different edge genes. Transcriptional expression profiles responsive to Cd illustrated a dramatic elevation of Sa14F190 and Sa18F186 genes. Heterologous expression in a Cd-sensitive yeast cell line, we confirmed the functions of Sa14F190 gene encoding ABCC in Cd accumulation. Our study performed a comprehensive analysis of ABCCs in S. alfredii Hance, firstly mapped their tissue-specific expression patterns responsive to Cd stress, and characterized the roles of Sa14F190 genes in Cd accumulation.

Asperosaponin VI promotes bone marrow stromal cell osteogenic differentiation through the PI3K/AKT signaling pathway in an osteoporosis model.

Asperosaponin VI (ASA VI), a natural compound isolated from the well-known traditional Chinese herb Radix Dipsaci, has an important role in promoting osteoblast formation. However, its effects on osteoblasts in the context of osteoporosis is unknown. This study aimed to investigate the effects and mechanism of ASA VI action on the proliferation and osteogenic differentiation of bone marrow stromal cells isolated from the ovariectomized rats (OVX rBMSCs). The toxicity of ASA VI and its effects on the proliferation of OVX rBMSCs were measured using a CCK-8 assay. Various osteogenic differentiation markers were also analyzed, such as ALP activity, calcified nodule formation, and the expression of osteogenic genes, i.e., ALP, OCN, COL 1 and RUNX2. The results indicated that ASA VI promoted the proliferation of OVX rBMSCs and enhanced ALP activity and calcified nodule formation. In addition, while ASA VI enhanced the expression of ALP, OCN, Col 1 and RUNX2, treatment with LY294002 reduced all of these osteogenic effects and reduced the p-AKT levels induced by ASA VI. These results suggest that ASA VI promotes the osteogenic differentiation of OVX rBMSCs by acting on the phosphatidylinositol-3 kinase/AKT signaling pathway.

Functional analysis of the TMPRSS2:ERG fusion gene in cisplatin­induced cell death.

The TMPRSS2:E­twenty­six (ETS) gene fusion occurs frequently in a high proportion of patients with prostate cancer (PCa) in Western countries, and the aberrant expression of TMPRSS2: v­ETS avian erythroblastosis virus E26 oncogene homolog (ERG), the most common form of the corresponding protein, can regulate cell migration and contribute to tumor invasion and metastasis. However, its association with other cellular events, and in particular, cell death, remain unknown. To examine the function of such fusion genes, an expression plasmid containing the TMPRSS2:ERG (T1/E5) sequence (ΔERG) from a patient sample was constructed and transiently transfected into DU145 cells, which do not express the fusion gene. It was found that the overexpression of ΔERG significantly inhibited the ability of cisplatin to induce apoptosis in DU145 cells. By contrast, VCaP cells, which do contain TMPRSS2:ERG, were sensitized to cisplatin­induced apoptosis through siRNA inhibition of the fusion gene. To elucidate the underlying mechanism, a stable cell line expressing the ΔERG gene was constructed. Expression of ΔERG did not affect cell migration, but did protect cells from DNA damage and apoptosis induced by cisplatin. Furthermore, knockdown of ΔERG by short interfering RNA resulted in cells regaining their sensitivity to cisplatin. Finally, the gene coding for activating transcription factor 5, which is important for cell survival, may be upregulated by ΔERG. Taken together, these data point to a new function of the TMPRSS2:ERG fusion gene in regulating the apoptotic pathway.

Depletion of Paraspeckle Protein 1 Enhances Methyl Methanesulfonate-Induced Apoptosis through Mitotic Catastrophe.

Previously, we have shown that paraspeckle protein 1 (PSPC1), a protein component of paraspeckles that was involved in cisplatin-induced DNA damage response (DDR), probably functions at the G1/S checkpoint. In the current study, we further examined the role of PSPC1 in another DNA-damaging agent, methyl methanesulfonate (MMS)-induced DDR, in particular, focusing on MMS-induced apoptosis in HeLa cells. First, it was found that MMS treatment induced the expression of PSPC1. While MMS treatment alone can induce apoptosis, depletion of PSPC1 expression using siRNA significantly increased the level of apoptosis following MMS exposure. In contrast, overexpressing PSPC1 decreased the number of apoptotic cells. Interestingly, morphological observation revealed that many of the MMS-treated PSPC1-knockdown cells contained two or more nuclei, indicating the occurrence of mitotic catastrophe. Cell cycle analysis further showed that depletion of PSPC1 caused more cells entering the G2/M phase, a prerequisite of mitosis catastrophe. On the other hand, over-expressing PSPC1 led to more cells accumulating in the G1/S phase. Taken together, these observations suggest an important role for PSPC1 in MMS-induced DDR, and in particular, depletion of PSPC1 can enhance MMS-induced apoptosis through mitotic catastrophe.

Methyl methanesulfonate induces necroptosis in human lung adenoma A549 cells through the PIG-3-reactive oxygen species pathway.

Methyl methanesulfonate (MMS) is an alkylating agent that can induce cell death through apoptosis and necroptosis. The molecular mechanisms underlying MMS-induced apoptosis have been studied extensively; however, little is known about the mechanism for MMS-induced necroptosis. Therefore, we first established MMS-induced necroptosis model using human lung carcinoma A549 cells. It was found that, within a 24-h period, although MMS at concentrations of 50, 100, 200, 400, and 800 µM can induce DNA damage, only at higher concentrations (400 and 800 µM) MMS treatment lead to necroptosis in A549 cells, as it could be inhibited by the specific necroptotic inhibitor necrostatin-1, but not the specific apoptotic inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk). MMS-induced necroptosis was further confirmed by the induction of the necroptosis biomarkers including the depletion of cellular NADH and ATP and leakage of LDH. This necroptotic cell death was also concurrent with the increased expression of p53, p53-induced gene 3 (PIG-3), high mobility group box-1 protein (HMGB1), and receptor interaction protein kinase (RIP) but not the apoptosis-associated caspase-3 and caspase-9 proteins. Elevated reactive oxygen species (ROS) level was also involved in this process as the specific ROS inhibitor (4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC)) can inhibit the necroptotic cell death. Interestingly, knockdown of PIG-3 expression by small interfering RNA (siRNA) treatment can inhibit the generation of ROS. Taken together, these results suggest that MMS can induce necroptosis in A549 cells, probably through the PIG-3-ROS pathway.

Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase.

Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.

BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle.

Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the two prominent pathways responsible for the repair of DNA double-strand breaks (DSBs). NHEJ is not restricted to a cell-cycle stage, whereas HR is active primarily in the S/G2 phases suggesting there are cell cycle-specific mechanisms that play a role in the choice between NHEJ and HR. Here we show NHEJ is attenuated in S phase via modulation of the autophosphorylation status of the NHEJ factor DNA-PKcs at serine 2056 by the pro-HR factor BRCA1. BRCA1 interacts with DNA-PKcs in a cell cycle-regulated manner and this interaction is mediated by the tandem BRCT domain of BRCA1, but surprisingly in a phospho-independent manner. BRCA1 attenuates DNA-PKcs autophosphorylation via directly blocking the ability of DNA-PKcs to autophosphorylate. Subsequently, blocking autophosphorylation of DNA-PKcs at the serine 2056 phosphorylation cluster promotes HR-required DNA end processing and loading of HR factors to DSBs and is a possible mechanism by which BRCA1 promotes HR.

Paraspeckle protein 1 (PSPC1) is involved in the cisplatin induced DNA damage response--role in G1/S checkpoint.

Paraspeckle protein 1 (PSPC1) was first identified as a structural protein of the subnuclear structure termed paraspeckle. However, the exact physiological functions of PSPC1 are still largely unknown. Previously, using a proteomic approach, we have shown that exposure to cisplatin can induce PSPC1 expression in HeLa cells, indicating the possible involvement for PSPC1 in the DNA damage response (DDR). In the current study, the role of PSPC1 in DDR was examined. First, it was found that cisplatin treatment could indeed induce the expression of PSPC1 protein. Abolishing PSPC1 expression by siRNA significantly inhibited cell growth, caused spontaneous cell death, and increased DNA damage. However, PSPC1 did not co-localize with γH2AX, 53BP1, or Rad51, indicating no direct involvement in DNA repair pathways mediated by these molecules. Interestingly, knockdown of PSPC1 disrupted the normal cell cycle distribution, with more cells entering the G2/M phase. Furthermore, while cisplatin induced G1/S arrest in HeLa cells, knockdown of PSPC1 caused cells to escape the G1/S checkpoint and enter mitosis, and resulted in more cell death. Taken together, these observations indicate a new role for PSPC1 in maintaining genome integrity during the DDR, particularly in the G1/S checkpoint.