TRESK Regulates Gm11874 to Induce Apoptosis of Spinal Cord Neurons via ATP5i Mediated Oxidative Stress and DNA Damage.

Reportedly, TWIK-related spinal cord K+ (TRESK) deficiency in spinal cord neurons positively correlates with the mechanism underlying neuropathic pain (NP). However, the precise effects of TRESK on neurons of the spinal cord remain elusive. In the present study, we investigated the impact of TRESK silencing on spinal cord neurons to further elucidate the downstream mechanisms of TRESK. Herein, neurons of the dorsal spinal cord were cultured as a cell model for investigations. Apoptosis, oxidative stress, and DNA damage-related proteins were evaluated. Additionally, flow cytometry, microarray profiling, real-time polymerase chain reaction (PCR), western blotting, fluorescence in situ hybridization (FISH), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were performed. In cultured neurons, the downregulation of TRESK mRNA expression induced apoptosis of dorsal spinal cord neurons. Using real-time PCR and western blotting, the upregulation of LncRNA Gm11874 (Gm11874) and ATP5i, screened from the gene chip, was confirmed. On silencing TRESK, expression levels of γ-H2AX, poly [ADP-ribose] polymerase 1 (PARP-1), FoxO1, FoxO3, MitoSOX, malondialdehyde (MDA), and 8-hydroxy-2' -deoxyguanosine (8-OHdG), which are known indices of oxidative stress and DNA damage, were significantly elevated. Moreover, ATP induced oxidative stress, DNA damage, and apoptosis were reduced by ATP5i siRNA. Finally, Gm11874 and ATP5i were co-expressed in spinal cord neurons in a FISH experiment, and the expression of ATP5i was positively regulated by Gm11874. These results implied that ATP5i induced oxidative stress and DNA damage, resulting in neuronal apoptosis, and Gm11874 was confirmed to act upstream of ATP5i. Our study revealed that TRESK silencing upregulated Gm11874 to induce apoptosis of spinal cord neurons, which resulted in ATP5i promoting oxidative stress and DNA damage. These findings could highlight the TRESK-mediated NP mechanism.

Validation of novel hub genes and molecular mechanisms in acute lung injury using an integrative bioinformatics approach.

Acute lung injury (ALI) is an inflammatory pulmonary disease that can easily develop into serious acute respiratory distress syndrome, which has high morbidity and mortality. However, the molecular mechanism of ALI remains unclear, and few molecular biomarkers for diagnosis and treatment have been identified. In this study, we aimed to identify novel molecular biomarkers using a bioinformatics approach. Gene expression data were obtained from the Gene Expression Omnibus database, co-expressed differentially expressed genes (CoDEGs) were identified using R software, and further functional enrichment analyses were conducted using the online tool Database for Annotation, Visualization, and Integrated Discovery. A protein-protein interaction network was established using the STRING database and Cytoscape software. Lipopolysaccharide (LPS)-induced ALI mouse model was constructed and verified. The hub genes were screened and validated in vivo. The transcription factors (TFs) and miRNAs associated with the hub genes were predicted using the NetworkAnalyst database. In total, 71 CoDEGs were screened and found to be mainly involved in the cytokine-cytokine receptor interactions, and the tumor necrosis factor and malaria signaling pathways. Animal experiments showed that the lung injury score, bronchoalveolar lavage fluid protein concentration, and wet-to-dry weight ratio were higher in the LPS group than those in the control group. Real-time polymerase chain reaction analysis indicated that most of the hub genes such as colony-stimulating factor 2 (Csf2) were overexpressed in the LPS group. A total of 20 TFs including nuclear respiratory factor 1 (NRF1) and two miRNAs were predicted to be regulators of the hub genes. In summary, Csf2 may serve as a novel diagnostic and therapeutic target for ALI. NRF1 and mmu-mir-122-5p may be key regulators in the development of ALI.

A telomere-targeting drug depletes cancer initiating cells and promotes anti-tumor immunity in small cell lung cancer.

There are few effective treatments for small cell lung cancer (SCLC) underscoring the need for innovative therapeutic approaches. This study focuses on exploiting telomerase, a critical SCLC dependency as a therapeutic target. A prominent characteristic of SCLC is their reliance on telomerase activity, a key enzyme essential for their continuous proliferation. Here we utilize a nucleoside analog, 6-Thio-2'-deoxyguanosine (6TdG) currently in phase II clinical trials, that is preferentially incorporated by telomerase into telomeres leading to telomere dysfunction. Using preclinical mouse and human derived models we find low intermittent doses of 6TdG inhibit tumor growth and reduce metastatic burden. Anti-tumor efficacy correlates with a reduction in a subpopulation of cancer initiating like cells (CICs) identified by their expression of L1CAM/CD133 and highest telomerase activity. 6TdG treatment also leads to activation of innate and adaptive anti-tumor responses. Mechanistically, 6TdG depletes CICs and induces type-I interferon signaling leading to tumor immune visibility by activating tumor cell STING signaling. We also observe increased sensitivity to irradiation after 6TdG treatment in both syngeneic and humanized SCLC xenograft models both of which are dependent on the presence of host immune cells. This study underscores the immune-enhancing and metastasis-reducing effects of 6TdG, employing a range of complementary in vitro and in vivo SCLC preclinical models providing a potential therapeutic approach to SCLC.

Loss of p53 and mutational heterogeneity drives immune resistance in an autochthonous mouse lung cancer model with high tumor mutational burden.

The role of tumor mutational burden (TMB) in shaping tumor immunity is a key question that has not been addressable using genetically engineered mouse models (GEMMs) of lung cancer. To induce TMB in lung GEMMs, we expressed an ultra-mutator variant of DNA polymerase-E (POLE)P286R in lung epithelial cells. Introduction of PoleP286R allele into KrasG12D and KrasG12D; p53L/L (KP) models significantly increase their TMB. Immunogenicity and sensitivity to immune checkpoint blockade (ICB) induced by Pole is partially dependent on p53. Corroborating these observations, survival of NSCLC patients whose tumors have TP53truncating mutations is shorter than those with TP53WT with immunotherapy. Immune resistance is in part through reduced antigen presentation and in part due to mutational heterogeneity. Total STING protein levels are elevated in Pole mutated KP tumors creating a vulnerability. A stable polyvalent STING agonist or p53 induction increases sensitivity to immunotherapy offering therapeutic options in these polyclonal tumors.

Triiodothyronine attenuates neurocognitive dysfunction induced by sevoflurane in the developing brain of neonatal rats.

BACKGROUND: Whilst concerns have been raised about the detrimental effects of general anaesthetics on the brain's development and function in the young, reports have indicated that thyroid hormones are able to promote neurogenesis in the developing brain. This present study aimed to investigate the effects of triiodothyronine (T3) on the neonatal rat brain, following sevoflurane exposure. METHODS: Postnatal day 7 (P7) ratpups were treated with Triiodothyronine (T3) (1 µg/100 g body weight, i.p. injection, once/day for 3 days) after 2% sevoflurane exposure for 6 h. They were sacrificed at either P7 (immediately), P15 or P30 and their brains were harvested to assess cell death, proliferation in the hippocampus, N-methyl-D-aspartate (NMDA) receptor subunit A and B, and a post-synaptic protein (PSD-95 in the hippocampus,). Neuro-behavioral changes in other cohorts between P27 and P30 were evaluated with Morris water maze and open field tests. RESULTS: Sevoflurane exposure caused cell death and suppressed the proliferation of astrocytes and neurons, as well as the dendritic growth of neurons in the hippocampus which were all reversed by the administration of T3. Moreover, cognitive function, including learning, memory, and adaptability to a new environment, were impaired by sevoflurane exposure, which was also negated by T3 treatment. Furthermore, sevoflurane decreased the expression of NMDA receptor subunits NR2A and NR2B, as well as PSD-95 in the hippocampus at P15 and those effects of sevoflurane were abolished by T3 administration. CONCLUSIONS: A potential therapeutic role of T3 in protecting general anesthetic induced neuronal injury in the developing brain is likely to occur through enhancing expression of PSD-95 and the NMDA NR2A and NR2B expression.

Melatonin inhibits microglial activation, reduces pro-inflammatory cytokine levels, and rescues hippocampal neurons of adult rats with acute Klebsiella pneumoniae meningitis.

Acute bacterial meningitis caused by Klebsiella pneumoniae (K. pneumoniae) is a major health threat with a high mortality rate and severe neuro-cognitive sequelae. The intense pro-inflammatory cytokine released from calcium-mediated microglial activation plays an important role in eliciting neuronal damage in the hippocampal region. Considering melatonin possesses anti-inflammatory and immuno-modulatory properties, the present study determined whether melatonin can effectively decrease inflammatory responses and prevent hippocampal damage in animals subjected to K. pneumoniae. Adult rats inoculated with K. pneumoniae received a melatonin injection immediately thereafter at doses of 5, 25, 50, or 100 mg/kg. Following 24 h of survival, all experimental animals were processed for time-of-flight secondary ion mass spectrometry (for detecting glial calcium intensity), isolectin-B4 histochemistry (reliable marker for microglial activation), pro-inflammatory cytokine measurement as well as cytochrome oxidase and in situ dUTP end-labeling (representing neuronal bio-energetic status and apoptotic changes, respectively). Results indicate that in K. pneumoniae-infected rats, numerous calcium-enriched microglia, enhanced pro-inflammatory cytokine, and various apoptotic neurons with low bio-energetic activity were detected in hippocampus. Following melatonin administration, however, all parameters including glial calcium intensity, microglial activation, pro-inflammatory cytokine levels, and number of apoptotic neurons were successfully decreased with maximal change observed at a melatonin dose of 100 mg/kg. Enzymatic data corresponded well with above findings in which all surviving neurons displayed high bio-energetic activity. As effectively reducing glia-mediated inflammatory response is neuro-protective to hippocampal neurons, the present study supports the clinical use of melatonin as a potential therapeutic agent to counteract K. pneumoniae meningitis-induced neuro-cognitive damage.

MicroRNAs in the Spinal Microglia Serve Critical Roles in Neuropathic Pain.

Neuropathic pain (NP) can occur after peripheral nerve injury (PNI), and it can be converted into a maladaptive, detrimental phenotype that causes a long-term state of pain hypersensitivity. In the last decade, the discovery that dysfunctional microglia evoke pain, called "microgliopathic pain," has challenged traditional neuronal views of "pain" and has been extensively explored. Recent studies have shown that microRNAs (miRNAs) can act as activators or inhibitors of spinal microglia in NP conditions. We first briefly review spinal microglial activation in NP. We then comprehensively describe miRNA expression changes and their potential mechanisms in the response of microglia to nerve injury. We summarize the roles of the following two representative miRNAs: miR-124, which reverses NP by keeping microglia quiescent, and miR-155, which promotes NP following microglial activation. Finally, we focused on the therapeutic potential of microglial miRNAs in NP. The findings we summarized may be essential tools for basic research and clinical treatment of NP.

miR-126 targeting GOLPH3 inhibits the epithelial-mesenchymal transition of gastric cancer BGC-823 cells and reduces cell invasion.

The incidence and mortality of gastric cancer have been increasing in recent years. MiR-126 and target genes have been studied in gastric cancer, but their studies with Golgi phosphoprotein 3 (GOLPH3) and related pathways in gastric cancer are rarely reported. In the present study, we aimed to investigate the interaction between the miR-126 and GOLPH3in the progression of gastric cancer. In this study, we revealed the role of miR-126-GOLPH3 axis into regulating the progression of epithelial-mesenchymal transition (EMT) in BGC-823 cell model. Firstly, tumor tissues and adjacent normal tissues were collected from 45 patients with gastric cancer. We found the expression of miR-126 in human tumor tissue was significantly lower than in normal tissue using reverse transcription-polymerase chain reaction (RT-PCR). But the GOLPH3 expression was opposite by the detection of immunohistochemistry, RT-PCR and Western blot. Moreover, we predicted miR-126 targeting GOLPH3 by bioinformatics and confirmed the interaction using luciferase reporter gene system; miR-126 inhibited the proliferation, invasion and EMT progression in BGC-823 cells through overexpressing miR-126; miR-126 negative regulated GOLPH3 expression by overexpressing and interfering miR-126. Finally, we found GOLPH3 could promote proliferation using MTT assay, invasion using Transwell, and EMT progression by inhibiting the expression of E-cadherin, inducing vimentin and N-cadherin in BGC-823 cells. Our results demonstrated that miR-126 inhibits proliferative and invasive ability as well as EMT progression by targeting GOLPH3. This study may provide a new field of vision for targeted treatment of gastric cancer.

Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats.

Autophagy maintains cellular homeostasis by stimulating the lysosomal degradation of cytoplasmic structures, including damaged organelles and dysfunctional proteins. The role of autophagy in the renewal and regeneration of injured peripheral nerves remains poorly understood. The current study investigated the role of autophagy in peripheral nerve regeneration and motor function recovery following sciatic nerve crush injury in rats by stimulating or suppressing autophagy and detecting the presence of autophagosomes and LC3-II expression by electron microscopy and Western blotting, respectively. Neurobehavioral function was tested by CatWalk gait analysis 1, 2, 3, and 6 weeks after injury, and the expression of neurofilament (NF)-200 and myelin basic protein (MBP) at the injury site was examined by immunocytochemistry. Apoptosis at the lesion site was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Treatment of injured rats with the autophagy inducer rapamycin increased the number of autophagosomes and LC3-II expression while reducing the number of apoptotic cells at the lesion; this was associated with an upregulation of MBP and NF-200 expression and increased motor function recovery as compared to sham-operated rats and those that were subjected to crush injury but untreated. The opposite effects were observed in rats treated with the autophagy inhibitor 3-methyladenine. These data indicate that the modulation of autophagy in peripheral nerve injury could be an effective pharmacological approach to promote nerve regeneration and reestablish motor function.