Several ribosomal proteins regulate p53 function by modulating MDM2. We recently found that RPS27L, a RPS27-like protein, is a direct p53-inducible target. Here we showed that RPS27 itself is a p53-repressible target. Furthermore, the N-terminal region of either RPS27L or RPS27 binds to MDM2 on the central acidic domain of MDM2. RPS27L or RPS27 forms an in vivo triplex with MDM2-p53 and competes with p53 for MDM2 binding. Similar to p53, RPS27L, but not RPS27, is a short-lived protein and a novel MDM2 substrate. Degradation of RPS27L requires the RING or acidic domain of MDM2. Ectopic expression of RPS27L or RPS27 inhibits MDM-2-mediated p53 ubiquitination and increases p53 levels by extending p53 protein half-life, whereas siRNA silencing of RPS27L decreases p53 levels by shortening p53 half-life, with a corresponding reduction in p53 transcription activity. RPS27L is mainly localized in the cytoplasm, but upon p53-activating signals, a portion of RPS27L shuttled to the nucleoplasm where it colocalizes with MDM2. Both the cytoplasmic and the nuclear p53, induced by ribosomal stress, were reduced upon RPS27L silencing. Our study reveals a multilevel interplay between RPS27L/S27 and p53-MDM2 axis, with RPS27L functioning as a p53 target, a MDM2 substrate and a p53 regulator.

It has been reported that some single nucleotide polymorphisms (SNPs) of the angiotensin converting enzyme (ACE) gene and the endothelial nitric oxide synthase (eNOS) gene are associated with the development of systemic lupus erythematosus (SLE) and the progression of nephropathy. The aim of this study was to evaluate the possible association between six SNPs (A-5466C, T-3892C, A-240T, C1237T, G2215A and A2350G) of the ACE gene and two SNPs (T-786C and G894T) of the eNOS gene with lupus nephropathy in a northern Chinese population.

Estradiol and progesterone bind to their respective receptors in the hypothalamus and hippocampus to influence a variety of behavioral and physiological functions, including reproduction and cognition. Work from our lab and others has shown that the nuclear receptor coactivators, steroid receptor coactivator-1 (SRC-1) and SRC-2, are essential for efficient estrogen receptor (ER) and progestin receptor (PR) transcriptional activity in brain and for hormone-dependent behaviors. While the expression of SRC-1 in brain has been studied extensively, little is known about the expression of SRC-2 in brain. In the present studies, we found that SRC-2 was highly expressed throughout the hippocampus, amygdala and hypothalamus, including the medial preoptic area (MPOA), ventral medial nucleus (VMN), arcuate nucleus (ARC), bed nucleus of the stria terminalis, supraoptic nucleus and suprachiasmatic nucleus. In order for coactivators to function with steroid receptors, they must be expressed in the same cells. Indeed, SRC-2 and ER(alpha) were coexpressed in many cells in the MPOA, VMN and ARC, all brain regions known to be involved in female reproductive behavior and physiology. While in vitro studies indicate that SRC-2 physically associates with ER and PR, very little is known about receptor-coactivator interactions in brain. Therefore, we used pull-down assays to test the hypotheses that SRC-2 from hypothalamic and hippocampal tissue physically associate with ER and PR subtypes in a ligand-dependent manner. SRC-2 from both brain regions interacted with ER(alpha) bound to agonist, but not in the absence of ligand or in the presence of the selective ER modulator, tamoxifen. Analysis by mass spectrometry confirmed these ligand-dependent interactions between ER(alpha) and SRC-2 from brain. In dramatic contrast, SRC-2 from brain showed little to no interaction with ERbeta. Interestingly, SRC-2 from both brain regions interacted with PR-B, but not PR-A, in a ligand-dependent manner. Taken together, these findings reveal that SRC-2 is expressed in brain regions known to mediate a variety of steroid-dependent functions. Furthermore, SRC-2 is expressed in many ER(alpha) containing cells in the hypothalamus. Finally, SRC-2 from brain interacts with ER and PR in a subtype-specific manner, which may contribute to the functional differences of these steroid receptor subtypes in brain.

Vitamin D has potential benefits for extraskeletal health. These could include an anti-inflammatory effect as well as a reduction in endothelial dysfunction. We aim to provide quality evidence for the hypothesis that supplementation with vitamin D will improve endothelial function (EF), possibly through the abrogation of systemic inflammation.

Cancer cells have distinct metabolomic profile. Metabolic enzymes regulate key oncogenic signaling pathways and have an essential role on tumor progression. Here, serum metabolomic analysis was performed in 45 patients with T-cell lymphoma (TCL) and 50 healthy volunteers. The results showed that dysregulation of choline metabolism occurred in TCL and was related to tumor cell overexpression of choline kinase-α (Chokα). In T-lymphoma cells, pharmacological and molecular silencing of Chokα significantly decreased Ras-GTP activity, AKT and ERK phosphorylation and MYC oncoprotein expression, leading to restoration of choline metabolites and induction of tumor cell apoptosis/necropotosis. In a T-lymphoma xenograft murine model, Chokα inhibitor CK37 remarkably retarded tumor growth, suppressed Ras-AKT/ERK signaling, increased lysophosphatidylcholine levels and induced in situ cell apoptosis/necropotosis. Collectively, as a regulatory gene of aberrant choline metabolism, Chokα possessed oncogenic activity and could be a potential therapeutic target in TCL, as well as other hematological malignancies with interrupted Ras signaling pathways.

We have previously reported that presynaptic dysfunction and cognitive decline have been found in lipoprotein lipase (LPL) deficient mice, but the mechanism remains to be elucidated. Accumulating evidence supported that α-synuclein (α-syn) and ubiquitin C-terminal hydrolase L1 (UCHL1) are required for normal synaptic and cognitive function. In this study, we found that α-syn aggregated and the expression of UCHL1 decreased in the brain of LPL deficient mice. Reduction of UCHL1 was resulted from nuclear retention of DNA cytosine-5-methyltransferase 1 in LPL knockout mice. Reverse changes were found in cultured cells overexpressing LPL. Furthermore, deficiency of LPL increased ubiquitination of α-syn. These results indicated that aggregation of α-syn and reduction of UCHL1 expression in LPL-deficient mice may affect synaptic function.

Chronic infection, such as Helicobacter pylori infection, has been associated with the development of gastric cancer (GC). Pathogen-associated molecular patterns can trigger inflammatory responses via Toll-like receptors (TLRs) in GC. Here we showed that Toll-like receptor 4 (TLR4) was highly expressed in GC cells and was associated with the aggressiveness of GC. The binding of lipopolysaccharide (LPS) to TLR4 on GC cells enhanced proliferation without affecting apoptosis. Higher level of reactive oxygen species (ROS) was induced after activation of TLR4 signaling in GC. Using oxidase inhibitors and antioxidants, we found that mitochondrial ROS (mROS) was major source of TLR4-stimulated ROS generation. This elevated mROS production can be inhibited by diphenylene iodonium (DPI), and the blocking of the mROS production rather than ROS neutralization resulted in cell cycle arrest and the loss of mitochondrial potential, which were plausible reason for decreased cell viability. Furthermore, the increased mROS owing to TLR4 signaling resulted in the activation of Akt phosphorylation and NF-κB p65 nuclear translocation. Altogether, these results reveal a novel pathway linking innate immune signaling to GC cell proliferation, implicate mROS as an important component of cell survival signals and further establish mitochondria as hubs for GC therapies.

Traumatic brain injury (TBI) produces excessive glutamate, leading to excitotoxicity via the activation of glutamate receptors. Postsynaptic density scaffold proteins have crucial roles in mediating signal transduction from glutamate receptors to their downstream mediators. Therefore, studies on the mechanisms underlying regulation of excitotoxicity by scaffold proteins can uncover new treatments for TBI. Here, we demonstrated that the postsynaptic scaffold protein Homer 1a was neuroprotective against TBI in vitro and in vivo, and this neuroprotection was associated with its effects on group I metabotropic glutamate receptors (mGluRs). Upon further study, we found that Homer 1a mainly affected neuronal injury induced by mGluR1 activation after TBI and also influenced mGluR5 function when its activity was restored. The ability of Homer 1a to disrupt mGluR-ERK signaling contributed to its ability to regulate the functions of mGluR1 and mGluR5 after traumatic injury. Intracellular Ca(2+) and PKC were two important factors involved in the mediation of mGluR-ERK signaling by Homer 1a. These results define Homer 1a as a novel endogenous neuroprotective agent against TBI.

Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.

Mesenchymal stromal cells (MSCs) are heterogeneous cells with immunoregulatory and wound-healing properties. In cancer, they are known to be an essential part of the tumour microenvironment. However, their role in tumour growth and rejection remains unclear. To investigate this, we co-cultured human MSCs, tumour infiltrating lymphocytes (TIL), and melanoma cells to investigate the role of MSCs in the tumour environment.

Bcl-2-like members have been found to be inherently overexpressed in many types of haematologic malignancies. The small-molecule S1 is a BH3 mimetic and a triple inhibitor of Bcl-2, Mcl-1 and Bcl-XL.

CREB-binding protein (CBP) and p300 are highly homologous transcriptional coactivators with unique, non-redundant roles that bind a wide array of proteins, including catenins-β and γ. ICG-001 is a small-molecule inhibitor that specifically inhibits the CBP/catenin interaction. Importantly, ICG-001 does not inhibit the p300/catenin interaction. We demonstrate that specifically inhibiting the interaction between CBP and catenin with ICG-001 results in the differentiation of quiescent drug-resistant chronic myelogenous leukemia-initiating cells (CML LICs), thereby sensitizing them to BCR-ABL tyrosine kinase inhibitors, for example, Imatinib. Using ICG-001 in a NOD/SCID/IL2Rγ(-/-) mouse model of engrafted human chronic myelogenous leukemia, we now demonstrate the complete elimination of engrafted leukemia after only one course of combined chemotherapy. Combination-treated animals live as long as their non-engrafted littermates. Results from these studies demonstrate that specifically antagonizing the CBP/catenin interaction with ICG-001 can eliminate drug-resistant CML LICs without deleterious effects to the normal endogenous hematopoietic stem cell population.

Osteonecrosis of the jaws is recognised as a serious complication for patients receiving bisphosphonates. The anti-angiogenic effects of bisphosphonates have been implicated in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The purpose of this study was to determine the effects of zoledronic acid on cultured human gingival fibroblasts in relation to the modulation of genes associated with angiogenic regulation.

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Despite the impressive clinical efficacy of T cells engineered to express chimeric antigen receptors (CAR-Ts), the current applications of CAR-T cell therapy are limited by major treatment-related toxicity. Thus, safer yet effective alternative approaches must be developed. In this study, we compared CD19 bispecific T-cell engager (BiTE)-transferred T cells that had been transfected by RNA electroporation with CD19 CAR RNA-transferred T cells both in vitro and in an aggressive Nalm6 leukemia mouse model. BiTEs were secreted from the transferred T cells and enabled both the transferred and bystander T cells to specifically recognize CD19(+) cell lines, with increased tumor killing ability, prolonged functional persistence, increased cytokine production and potent proliferation compared with the CAR-T cells. More interestingly, in comparison with CD3/CD28 bead-stimulated T cells, T cells that were expanded by a rapid T-cell expansion protocol (REP) showed enhanced anti-tumor activities for both CAR and BiTE RNA-electroporated T cells both in vitro and in a Nalm6 mouse model (P<0.01). Furthermore, the REP T cells with BiTE RNAs showed greater efficacy in the Nalm6 leukemia model compared with REP T cells with CAR RNA (P<0.05) and resulted in complete leukemia remission.

A number of experimental investigations utilizing different murine species have previously reported: (i) that standard mouse-diets supplemented with physiologically realistic amounts of neurotoxic metal salts substantially induce pro-inflammatory signaling in a number of murine tissues; (ii) that these diet-stimulated changes may contribute to a systemic inflammation (SI), a potential precursor to neurodegenerative events in both the central and the peripheral nervous system (CNS, PNS); and (iii) that these events may ultimately contribute to a chronic and progressive inflammatory neurodegeneration, such as that which is observed in Alzheimer's disease (AD) brain. In these experiments we assayed for markers of SI in the blood serum of C57BL/6J mice after 0, 1, 3 and 5 months of exposure to a standard mouse diet that included aluminum-sulfate in the food and drinking water, compared to age-matched controls receiving magnesium-sulfate or no additions. The data indicate that the SI markers that include the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), the acute phase reactive protein C-reactive protein (CRP) production and a triad of pro-inflammatory microRNAs (miRNA-9, miRNA-125b and miRNA-146a) all increase in the serum after aluminum-sulfate exposure. For the first time these results suggest that ad libitum exposure to aluminum-sulfate at physiologically realistic concentrations, as would be found in the human diet over the long term, may predispose to SI and the potential development of chronic, progressive, inflammatory neurodegeneration with downstream pathogenic consequences.

Klebsiella pneumoniae has been reported to develop increased antibiotic resistance. Ceftazidime-avibactam (CZA) is a novel antibiotic with activity against serine-lactamase. Here, we investigated the sensitivity of carbapenem-resistant K. pneumoniae (CRKP) to CZA and the mechanisms of drug resistance in our hospital.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

Recent studies indicate that the vasorelaxation induced by nitrendipine may be mediated partially by increased release of nitric oxide (NO). To study this effect in more detail and to examine the vasodilating effect of other 1,4-dihydropyridines (1,4-DHP) with regard to a possible involvement of NO, we investigated the effects of nitrendipine, nifedipine, nisoldipine, and nimodipine on isolated mesenteric vascular bed an the influence of L-NG-nitroarginine (L-NNA) on 1,4-DHP-induced vasorelaxation. Perfusion with these 1,4-DHP resulted in a concentration-dependent increase in global flow and vascular diameter in all vessel branches. Nifedipine exhibited a more pronounced effect on G4 vessels, whereas the actions of the other 1,4-DHP on the investigated vascular tree were more homogeneous. The dilating and flow increasing effects of nitrendipine and nifedipine could be significantly antagonized by treatment with LNNA. The vasodilating effects of nisoldipine and nimodipine could also be antagonized with L-NNA. We conclude that NO release plays an additional role in the relaxation of small resistance vessels by 1,4-DHP.

Neurofilament proteins of mammalian axotomized peripheral axons, which regenerate effectively, resemble those of embryonic axons. However, injured centrally projecting mammalian axons, which fail to regenerate, have very different neurofilament compositions than during development. If changes in neurofilament composition after injury reflect the ability of axotomized neurons to regenerate effectively, then the neurofilaments of centrally projecting axons that can regenerate should more closely resemble those of developing axons. In this study, the neurofilament compositions of injured optic axons of the frog, Xenopus laevis, were examined, since these axons can regenerate a fully functional projection. Antibodies to phosphorylated and nonphosphorylated forms of neurofilament proteins that had been used previously to study the neurofilament composition of newly developing X. laevis optic axons were used in immunocytochemical studies to examine the return of neurofilaments to the optic nerve after an intraorbital nerve crush. Intraocularly injected wheat germ agglutinin conjugated to horseradish peroxidase was used to label the regenerating axons independently of their neurofilaments. Neurofilament immunoreactivities disappeared rapidly from crushed axons during the first week after surgery. By nine days after surgery, antibodies to nonphosphorylated forms of middle (NF-M) and low molecular weight (NF-L) neurofilament proteins and the Xenopus neuronal intermediate filament protein (XNIF) began to stain the nerve just beyond the lesion. By this time, however, growing axonal terminals had reached the optic chiasm. Antibodies to phosphorylated epitopes of NF-M began to stain axons at 15 days, just as growing axons began to arrive at the optic tectum. Nonphosphorylated high molecular weight neurofilament protein (NF-H) began to appear in axons between 18 and 21 days after surgery. Thus, the reappearance of neurofilaments during optic axon regeneration resembled the general pattern seen during development. The chief difference between development and regeneration was that neurofilament epitopes took longer to emerge during regeneration. One possibility is that cues encountered along the optic pathway influence the neurofilament composition of retinal ganglion cell axons. Then, the greater distances travelled by regenerating axons could account for the longer time taken for their neurofilament compositions to mature.