8-Oxoguanine DNA glycosylase protects cells from senescence via the p53-p21 pathway.

Cellular senescence is an important factor leading to pulmonary fibrosis. Deficiency of 8-oxoguanine DNA glycosylase (OGG1) in mice leads to alleviation of bleomycin (BLM)-induced mouse pulmonary fibrosis, and inhibition of the OGG1 enzyme reduces the epithelial mesenchymal transition (EMT) in lung cells. In the present study, we find decreased expression of OGG1 in aged mice and BLM-induced cell senescence. In addition, a decrease in OGG1 expression results in cell senescence, such as increases in the percentage of SA-ß-gal-positive cells, and in the p21 and p-H2AX protein levels in response to BLM in lung cells. Furthermore, OGG1 promotes cell transformation in A549 cells in the presence of BLM. We also find that OGG1 siRNA impedes cell cycle progression and inhibits the levels of telomerase reverse transcriptase (TERT) and LaminB1 in BLM-treated lung cells. The increase in OGG1 expression results in the opposite phenomenon. The mRNA levels of senescence-associated secretory phenotype (SASP) components, including IL-1α, IL-1ß, IL-6, IL-8, CXCL1/CXCL2, and MMP-3, in the absence of OGG1 are obviously increased in A549 cells treated with BLM. Interestingly, we demonstrate that OGG1 binds to p53 to inhibit the activation of p53 and that silencing of p53 reverses the inhibition of OGG1 on senescence in lung cells. Additionally, the augmented cell senescence is shown in vivo in OGG1-deficient mice. Overall, we provide direct evidence in vivo and in vitro that OGG1 plays an important role in protecting tissue cells against aging associated with the p53 pathway.

Echocardiographic evaluation of right heart failure which might be associated with DNA damage response in SU5416-hypoxia induced pulmonary hypertension rat model.

Right heart failure is the leading cause of death in pulmonary hypertension (PH), and echocardiography is a commonly used tool for evaluating the risk hierarchy of PH. However, few studies have explored the dynamic changes in the structural and functional changes of the right heart during the process of PH. Previous studies have found that pulmonary circulation coupling right ventricular adaptation depends on the degree of pressure overload and other factors. In this study, we performed a time-dependent evaluation of right heart functional changes using transthoracic echocardiography in a SU5416 plus hypoxia (SuHx)-induced PH rat model. Rats were examined in 1-, 2-, 4-, and 6-week using right-heart catheterization, cardiac echocardiography, and harvested heart tissue. Our study found that echocardiographic measures of the right ventricle (RV) gradually worsened with the increase of right ventricular systolic pressure, and right heart hypofunction occurred at an earlier stage than pulmonary artery thickening during the development of PH. Furthermore, sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2), a marker of myocardial damage, was highly expressed in week 2 of SuHx-induced PH and had higher levels of expression of γ-H2AX at all timepoints, as well as higher levels of DDR-related proteins p-ATM and p53/p-p53 and p21 in week 4 and week 6. Our study demonstrates that the structure and function of the RV begin to deteriorate with DNA damage and cellular senescence during the early stages of PH development.

Diuretic-induced hypokalaemia: an updated review.

Diuretic-induced hypokalaemia is a common and potentially life-threatening adverse drug reaction in clinical practice. Previous studies revealed a prevalence of 7%-56% of hypokalaemia in patients taking thiazide diuretics. The clinical manifestations of hypokalaemia due to diuretics are non-specific, varying from asymptomatic to fatal arrhythmia. Diagnosis of hypokalaemia is based on the level of serum potassium. ECG is useful in identifying the more severe consequences. A high dosage of diuretics and concomitant use of other drugs that increase the risk of potassium depletion or cardiac arrhythmias can increase the risk of cardiovascular events and mortality. Thiazide-induced potassium depletion may cause dysglycaemia. The risk of thiazide-induced hypokalaemia is higher in women and in black people. Reducing diuretic dose and potassium supplementation are the most direct and effective therapies for hypokalaemia. Combining with a potassium-sparing diuretic or blocker of the renin-angiotensin system also reduces the risk of hypokalaemia. Lowering salt intake and increasing intake of vegetables and fruits help to reduce blood pressure as well as prevent hypokalaemia.

Evolution trace of SARS-CoV-2 from January 19 to March 12, 2020, in the United States.

As a kind of human betacoronavirus, SARS-CoV-2 has endangered globally public health. As of January 2021, the virus had resulted in 2,209,195 deaths. By studying the evolution trend and characteristics of 265 SARS-CoV-2 strains in the United States from January to March, it is found that the strains can be divided into six clades, USA clade-1, USA clade-2, USA clade-3, USA clade-4, USA clade-5, and USA clade-6, in which US clade-1 may be the most ancestral clade, USA clade-2 is an interim clade of USA clade-1 and USA clade-3, the other three clades have similar codon usage pattern, while USA clade-6 is the newest and most adaptable clade. Mismatch analysis and protein alignment showed that the evolution of the clades arises from some special mutations in viral proteins, which may help the strain to invade, replicate, transcribe and so on. Compared with previous research and classifications, we suggest that clade O in GISAID should not be an independent clade and Wuhan-Hu-1 (EPI_ISL_402125) should not be an ancestral reference sequence. Our study decoded the evolutionary dynamic of SARS-CoV-2 in the early stage from the United States, which give some clues to infer the current evolution trend of SARS-CoV-2 and study the function of viral mutational protein.

PTEN loss correlates with T cell exclusion across human cancers.

BACKGROUND: Recent evidences had shown that loss in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was associated with immunotherapy resistance, which may be attributed to the non-T-cell-inflamed tumor microenvironment. The impact of PTEN loss on tumor microenvironment, especially regarding T cell infiltration across tumor types is not well understood. METHODS: Utilizing The Cancer Genome Atlas (TCGA) and publicly available dataset of immunotherapy, we explored the correlation of PTEN expressing level or genomic loss with tumor immune microenvironment and response to immunotherapy. We further investigated the involvement of PI3K-AKT-mTOR pathway activation, which is known to be the subsequent effect of PTEN loss, in the immune microenvironment modulation. RESULTS: We reveal that PTEN mRNA expression is significantly positively correlated with CD4/CD8A gene expression and T cells infiltration especially T helpers cells, central memory T cell and effector memory T cells in multiples tumor types. Genomic loss of PTEN is associated with reduced CD8+ T cells, type 1 T helper cells, and increased type 2 T helper cells, immunosuppressed genes (e.g. VEGFA) expression. Furthermore, T cell exclusive phenotype is also observed in tumor with PI3K pathway activation or genomic gain in PIK3CA or PIK3CB. PTEN loss and PI3K pathway activation correlate with immunosuppressive microenvironment, especially in terms of T cell exclusion. PTEN loss predict poor therapeutic response and worse survival outcome in patients receiving immunotherapy. CONCLUSION: These data brings insight into the role of PTEN loss in T cell exclusion and immunotherapy resistance, and inspires further research on immune modulating strategy to augment immunotherapy.

8-Oxoguanine DNA glycosylase modulates the cell transformation process in pulmonary fibrosis by inhibiting Smad2/3 and interacting with Smad7.

The DNA repair enzyme 8-oxoguanine DNA glycosylase-1 (OGG1) is involved in early embryonic development, as well as in multiple conditions, including cardiac fibrosis, diabetes, and neurodegenerative diseases. But, function of OGG1 in pulmonary fibrosis was not entirely clear. In this study, we identified a novel function of OGG1 in the cell transformation process in pulmonary fibrosis. We demonstrated that OGG1 and Smad7 co-localize and interact in A549 cells. Bleomycin-induced pulmonary fibrosis was established in wild-type (WT) and Ogg1-/- mice. Upon treatment with transforming growth factor (TGF)-ß1, increased OGG1 expression was observed in WT mice with pulmonary fibrosis as well as in A549 cells, MRC-5 cells, and primary rat type II alveolar epithelial cells. The increased expression of OGG1 promoted cell migration, while OGG1 depletion decreased migration ability. Expression of the transformation-associated markers vimentin and alpha-smooth muscle actin were also affected by OGG1. We also observed that OGG1 promoted TGF-ß1-induced cell transformation and activated Smad2/3 by interacting with Smad7. The interaction between OGG1 and the TGF-ß/Smad axis modulates the cell transformation process in lung epithelial cells and fibroblasts. Moreover, we demonstrated that Ogg1 deficiency relieved pulmonary fibrosis in bleomycin-treated mice. Ogg1 knockout decreased the bleomycin-induced expression of Smad7 and phosphorylation of Smad2/3 in mice. These findings suggest that OGG1 has multiple biological functions in the pathogenesis of pulmonary fibrosis.

Risk factors associated with wheezing in severe pediatric community-acquired pneumonia: a retrospective study.

BACKGROUND: Wheezing is a common clinical manifestation in children with pneumonia. However, the risk factors associated with the development of wheezing pneumonia and its clinical features are not fully characterized, especially in children with severe pneumonia. METHODS: We retrospectively recruited 1434 pediatric patients diagnosed with severe pneumonia between April 2012 and September 2019 in Fujian Maternity and Child Health Hospital. The medical records regarding demographic information, clinical manifestations, radiographic/laboratory findings, and complications were collected. Based on the presence or absence of wheezing symptoms and signs, subjects were divided into wheezing cohort (n=684) and non-wheezing cohort (n=750), and their clinical data were compared. Multivariate cox regression analysis was performed to identify independent risk factors of wheezing. RESULTS: Demographic features including gender, weigh, onset season, birth weight, full-term birth or not, history of pneumonia were significantly associated with the occurrence of wheezing in severe CAP (P<0.05). Specifically, male gender, onset seasons in autumn/winter, and absence of a history of pneumonia were identified as independent risk factors of wheezing in multivariate analysis (P<0.05). As for clinical features, wheezing cohort differed from the non-wheezing one in terms of clinical manifestation (higher incidence of cough and breathless, but lower incidence of fever), laboratory finding (higher levels of red blood cells, hemoglobin, and albumin and lower levels of total or indirect bilirubin and creatine), pathogen detection (higher incidence of respiratory syncytial viral infection), and clinical complications (lesser risk of sepsis and hydrothorax) (P<0.05). CONCLUSIONS: Severe CAP with wheezing is a special clinical entity of severe pneumonia in children, which has specific risk factors and differ from non-wheezing pneumonia in terms of clinical features and etiologic pathogens.

A modified alternative fistula risk score (a-FRS) obtained from the computed tomography enhancement pattern of the pancreatic parenchyma predicts pancreatic fistula after pancreatoduodenectomy.

BACKGROUND: Alternative fistula risk score (a-FRS) is useful to predict clinically relevant postoperative pancreatic fistula (CR-POPF) after pancreatoduodenectomy (PD). METHODS: Clinical data from 239 patients undergoing PD were collected. The CT value of the pancreatic parenchyma was measured in the nonenhanced (N), arterial (A), portal venous (P), and late (L) phases. The A/N, A/P, P/L and A/L ratios were calculated and their correlation with CR-POPF were analyzed. By replacing pancreatic texture with the best CT attenuation ratio, a modified a-FRS was developed. RESULTS: Forty-seven patients developed CR-POPF. The A/P ratio (P < 0.001), P/L ratio (P = 0.002) and A/L ratio (P < 0.001) were significantly higher in the CR-POPF group. The A/L ratio performed best in predicting CR-POPF (AUC: 0.803) and the cut-off value is 1.36. A/L ratio >1.36 (P < 0.001), body mass index (P = 0.005) and duct diameter (P = 0.037) were independently associated with CR-POPF. By replacing soft texture with an A/L ratio >1.36, a modified a-FRS was developed and performed better than the a-FRS (AUC: 0.823 vs 0.748, P = 0.006) in predicting CR-POPF. CONCLUSIONS: The modified a-FRS is an objective and preoperative model for predicting the occurrence of CR-POPF after PD.

Structural and functional definition of the pulmonary vein system in a chronic hypoxia-induced pulmonary hypertension rat model.

Unlike the pulmonary artery (PA), the pathophysiological changes of the pulmonary vein (PV) in the development of pulmonary hypertension (PH) remain largely unknown. In this study, we comprehensively investigated the structural and functional changes in the PV isolated from the chronic hypoxia (CH; 10% O2, 21 days)-induced PH rat model (CHPH). Results showed that CH caused an increase in right ventricular pressure but did not affect the mean pulmonary venous pressure and the left atrial pressure. Similar to the PA, vascular lumen stenosis and medial thickening were also observed in the intrapulmonary veins isolated from the CHPH rats. Notably, CH induced more severe loss in the endothelium of intrapulmonary veins than the arteries. Then, the contractile response to 5-HT and U46619 was significantly greater in the intrapulmonary small veins (ISPV) and arteries (ISPA) isolated from CHPH rats than those from normoxic rats but not in the extrapulmonary and intrapulmonary large veins. Treatment with nifedipine (Nif), SKF96365 (SKF), or ryanodine and caffeine either partially attenuated (Nif) or dramatically abolished (SKF or ryanodine and caffeine) 5-HT-induced maximal contraction in ISPV from both normoxic and CHPH rats. Because of the severe loss of endothelium in the PV of CHPH rats, the decrease in acetylcholine (ACh)-induced endothelium-dependent relaxation was significantly larger in ISPV than ISPA, whereas the sodium nitroprusside-induced endothelium-independent relaxation was not altered in both ISPA and ISPV. In conclusion, our results provide fundamental data to comprehensively define the PV system in CHPH rat model.

Tetraspanin 1 as a mediator of fibrosis inhibits EMT process and Smad2/3 and beta-catenin pathway in human pulmonary fibrosis.

Tetraspanin 1(TSPAN1) as a clinically relevant gene target in cancer has been studied, but there is no direct in vivo or vitro evidence for pulmonary fibrosis (PF). Using reanalysing Gene Expression Omnibus data, here, we show for the first time that TSPAN1 was markedly down-regulated in lung tissue of patient with idiopathic PF (IPF) and verified the reduced protein expression of TSPAN1 in lung tissue samples of patient with IPF and bleomycin-induced PF mice. The expression of TSPAN1 was decreased and associated with transforming growth factor-ß1 (TGF-ß1 )-induced molecular characteristics of epithelial-to-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). Silencing TSPAN1 promoted cell migration, and the expression of alpha-smooth muscle actin, vimentin and E-cadherin in AECs with TGF-ß1 treatment, while exogenous TSPAN1 has the converse effects. Moreover, silencing TSPAN1 promotes the phosphorylation of Smad2/3 and stabilizes beta-catenin protein, however, overexpressed TSPAN1 impeded TGF-ß1 -induced activation of Smad2/3 and beta-catenin pathway in AECs. Together, our study implicates TSPAN1 as a key regulator in the process of EMT in AECs of IPF.

MiR-5100 increases the cisplatin resistance of the lung cancer stem cells by inhibiting the Rab6.

Cisplatin-based chemotherapy is the most commonly used treatment regimen for lung cancer. Cancer stem cells (CSCs) are postulated to be important promoters of drug resistance. We previously found that miR-5100 is overexpressed in lung cancer, but it is unknown whether and how miR-5100 regulates cisplatin resistance. Here, we demonstrated that miR-5100 was significantly up-regulated in CD44+ CD133+ lung cancer stem cells (LCSCs) compared with non-CSCs. Additionally, over-expression of miR-5100 increased CSC properties, cell growth, and tumor sphere formation in lung cancer cell line A549 or H1299, and that miR-5100 inhibitor significantly increased sensitivity of LCSCs to cisplatin in vitro. Surprisingly, the combination with miR-5100 inhibitor significantly decreased the IC50 of LCSCs to cisplatin. Furthermore, miR-5100 increased CSC properties and cisplatin resistance by inhibiting Rab6, a direct target gene of miR-5100. We demonstrated that miR-5100 overexpression increases the cisplatin resistance of the LCSCs through the mitochondrial apoptosis pathway. In conclusion, our results suggest that miR-5100 increases the cisplatin resistance of the LCSCs by inhibiting the Rab6. This study provides novel insight into the regulation of LCSCs by miRNA.

Tetraspanin 1 inhibits TNFα-induced apoptosis via NF-κB signaling pathway in alveolar epithelial cells.

OBJECTIVE: Tetraspanin family plays an important role in the pathogenesis of cancer, but its role in lung fibrosis is unknown. To determine whether tetraspanin 1 (TSPAN1), a member of the family, may be involved in the pathogenesis of pulmonary fibrosis. METHODS: TNFα -stimulated human alveolar epithelial (A549) and alveolar epithelial type II cell (AT2) were treated in vitro. Murine pulmonary fibrosis model was generated by injection of bleomycin (BLM). The expression of TSPAN1 was examined in vivo using the bleomycin-induced lung fibrosis model and tissue sample of IPF patients. Then we transfected the cells with TSPAN1 siRNA or plasmid and detected the expression changes of related proteins and cell apoptosis. RESULTS: In our study, we found that TSPAN1 was markedly down-regulated in lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and in bleomycin-induced pulmonary fibrosis in mice. We also found that TSPAN1 was significantly down-regulated in A549 and primary (AT2) cells following exposure to TNFα. Meanwhile, TSPAN1 inhibited p-IκBα, which attenuated nuclear NF-κB translocation and activation and inhibited apoptosis. We demonstrated that TSPAN1 reduced Bax translocation and caspase-3 activation, inhibited the apoptosis by regulating the NF-κB pathway in response to TNFα. CONCLUSIONS: We conclude that TSPAN1 mediated apoptosis resistance of alveolar epithelial cells by regulating the NF-κB pathway. TSPAN1 may be a potential therapeutic target for pulmonary fibrosis or acute lung injury.

Intrahepatic cholangiocarcinoma prognostic determination using pre-operative serum C-reactive protein levels.

BACKGROUND: Serum C-reactive protein (CRP), an acute inflammatory response biomarker, has been recognized as an indicator of malignant disease progression. However, the prognostic significance of CRP levels collected before tumor removal in intrahepatic cholangiocarcinoma requires further investigation. METHODS: We sampled the CRP levels in 140 patients with intrahepatic cholangiocarcinoma who underwent hepatectomies with regional lymphadenectomies between 2006 and 2013. A retrospective analysis of the clinicopathological data was performed. We focused on the impact of serum CRP on the patients' cancer-specific survival and recurrence-free survival rates. RESULTS: High levels of preoperative serum CRP were significantly associated with well-established clinicopathologic features, including gender, advanced tumor stage, and elevated carcinoembryonic antigen and carbohydrate antigen 19-9 levels (P < 0.05). Univariate analysis demonstrated a significant association between high levels of serum CRP and adverse cancer-specific survival (P = 0.001) and recurrence-free survival (P < 0.001). In patients with stage I/II intrahepatic cholangiocarcinoma, the serum CRP level was a prognostic indicator for cancer-specific survival. In patients with stage I/II or stage III/IV, the serum CRP level was a prognostic indicator for recurrence-free survival (P < 0.05). Additionally, multivariate analysis identified serum CRP level in intrahepatic cholangiocarcinoma as an independent prognostic factor (P < 0.05). CONCLUSIONS: We confirmed a significant association of elevated pre-operative CRP levels with poor clinical outcomes for the tested patients with intrahepatic cholangiocarcinoma. Our results indicate that the serum CRP level may represent a useful factor for patient stratification in intrahepatic cholangiocarcinoma management.

Abnormal miRNA-30e Expression is Associated with Breast Cancer Progression.

BACKGROUND: microRNAs (miRNAs) are involved in the regulation of various cellular processes, such as differentiation, proliferation, metabolism, and apoptosis, and they have been implicated in several diseases, including cancers. METHODS: To assess the role of miRNA in the progression of breast cancer, we performed TaqMan-based miRNA profiling for plasma from patients with breast cancer (n = 53), unrelated diseases (n = 40), or matched healthy controls (n = 40), and for breast tumors or adjacent non-tumors (n = 41). RESULTS: We selected 18 miRNAs with predicted roles in breast cancer and demonstrated that let-7i (p = 0.019), let-7a (p = 0.02), and miR-650 (p = 0.008) were significantly up-regulated in plasma; miR-21 (p < 0.001) is up-regulated in breast cancer tissue, and miR-30e was down-regulated in both plasma (p < 0.001) and breast cancer tissues (p = 0.004). Plasma miR-30e expression was shown to be statistically associated with age (p = 0.0402) and clinical stage (p = 0.007). However, receiver-operating characteristic curve analyses suggested that miR-30e expression cannot significantly differentiate breast cancer from healthy tissue or plasma. Consistent with a potential role for miR-30e in breast cancer, three predicted targets of miR-30e (RAB11A, BNIP3L, and RAB32) are up-regulated in breast cancer tissue. CONCLUSIONS: These findings suggest that reduced miR-30e correlates with the clinical stage of breast cancer. It is worthwhile to further explore that the potential role of miR-30e as a tumor suppressor in breast cancer, as well as its potential therapeutic utility.

Pro-inflammatory response and oxidative stress induced by specific components in ambient particulate matter in human bronchial epithelial cells.

Previous studies have shown that biological effect of particulate matter (PM2.5) is involved in including chemical composition and mass concentration, but the precise components and biological action on human bronchial epithelial cell line (BEAS-2B) are still unclear. The aim of this study was to evaluate the in vitro toxicity of PM2.5 collected at six urban sites in China, and to investigate how particle composition affects cytotoxicity. We used human bronchial epithelial (BEAS-2B) cell lines as model in vitro to expose to PM2.5 from different source, and then reactive oxygen species (ROS), superoxide dismutase activity and total antioxidant capacity were analyzed. Furthermore, we estimated the polycyclic aromatic hydrocarbon (PAH) and transition metal and the endotoxin contents. The mRNA expression of IL-1ß and IL-10 following exposure to PM2.5 was measured by QRT-PCR. We also observed the mitochondrial membrane potential (MMP) using JC-1 staining, and apoptosis of BEAS-2B using flow cytometry. In addition, double-stranded DNA breaks (DSBs) were assessed using γ-H2AX immunofluorescence. Our results show that high concentrations of PAHs and elemental Ni were strongly associated with high apoptosis rates and high expression of IL-1ß, in addition, Fe element was associated with the ROS level, furthermore, Fe and Cr element were associated with DNA damage in BEAS-2B cells. The cytotoxic effects of urban PM2.5 derived from six different cities in China appear dependent on the specific components in each. Our results indicate that air quality standards based on PM2.5 components may be more relevant than concentration-response functions (CRF). © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 923-936, 2016.

Mitochondrial OGG1 protects against PM2.5-induced oxidative DNA damage in BEAS-2B cells.

The enzyme 8-oxoguanine glycosylase 1 (OGG1) has been shown to be involved in the repair of oxidative DNA damage. However, the effect of OGG1 on oxidative DNA damage caused by particulate matter 2.5 (PM2.5) is unknown. Herein, we demonstrated that OGG1 could inhibit the generation of ROS and alleviate mitochondrial dysfunction and increased the expression of IL-1ß caused by PM2.5. The dichlorodihydrofluorescein diacetate (DCFH-DA) staining and 5,5',6,6'-tetrachloro-1,1',3,3'-. tetraethylbenzi-midazolylcarbocyanine iodide (JC-1) staining using flow cytometry showed that PM2.5 induces the generation of ROS and leads to a reduction in mitochondrial membrane potential (MMP) in BEAS-2B cells. Overexpression of OGG1 inhibited the generation of ROS and the decline in MMP. Knockdown of OGG1 by RNA interference (RNAi) increased the generation of ROS and reduced the MMP. Real-time quantitative PCR (RT-qPCR) for the mitochondrial DNA copy number (mtDNAcn) and flow cytometry for apoptosis revealed that OGG1 inhibits the apoptosis and decreases mtDNAcn induced by PM2.5. Additionally, the results of the comet assay showed that OGG1 had a significant repair effect on DNA strand breaks caused by PM2.5. Overexpression of OGG1 also significantly suppressed the expression of IL-1ß caused by PM2.5. Together, these data suggest that PM2.5 leads to mitochondrial dysfunction and the up-regulation of IL-1ß could be reversed by overexpression of OGG1. The mitochondrial dysfunction caused by PM2.5 could be relieved by OGG1. Thus, the base excision repair enzyme OGG1 may alleviate mitochondrial dysfunction caused by PM2.5 involved in the expression of IL-1ß.

Adenovirus-Mediated E2F-1 Gene Transfer Augments Gemcitabine-Induced Apoptosis in Human Colon Cancer Cells.

BACKGROUND: E2F-1 is a transcription factor that stimulates cellular proliferation and cell cycle progression. E2F-1 alone is sufficient to stimulate cells to initiate DNA synthesis, and this unscheduled entry into S phase is a potent trigger of apoptosis. Gemcitabine, a novel pyrimidine analogue with structural and metabolic similarities to cytarabine, also can efficiently induce apoptosis, especially for cancer cells that are already in S phase. Gemcitabine has established antitumor activity against solid tumors, including head and neck, ovarian, and non-small cell lung cancers. Therefore, we hypothesized that exogenous E2F-1 expression could accumulate cells in the S phase and thus sensitize them to gemcitabine. METHODS: We constructed an adenoviral vector (AdCMVE2F-1) to transduce the exogenous E2F-1 gene into human cancer cells. Infection of human colon cancer cells with AdCMVE2F-1 resulted in the overexpression of E2F-1 mRNA and protein in a dose-dependent manner and consequently induced accumulation in S phase as measured by FACS analysis. To assess the synergistic antitumor effect of AdCMVE2F-1 and gemcitabine, the human colon cancer cel lines SW620, DLD-1, and LoVo were infected with AdCMVE2F-1 at various multiplicities of infection and then exposed to various concentrations of gemcitabine 24 hours after infection. RESULT: Isobologram analysis showed that E2F-1-transduced cancer cells exhibited higher sensitivity to gemcitabine treatment compared to control virus-infected cells. Treatment with AdCMVE2F-1 plus gemcitabine enhanced endogenous p53 expression in LoVo cells, which contain wild-type p53; however, the finding that the synergistic effect can also be observed in mutant p53-expressing SW620 and DLD-1 cells suggests that wild-type p53 function may not be necessary for the therapeutic effects of this drug combination. Conclusions: Our data demonstrate that overexpression of ectopic E2F-1 protein may render cels more sensitive to gemcitabine-mediated apoptosis, an outcome that has important general implications for the treatment of human cancer.

Circulating microRNA Signatures Associated with Childhood Asthma.

BACKGROUND: MicroRNAs (miRNAs) exist stably and reproducibly in plasma and may be used as biomarkers for various diseases. Little is known about circulating miRNAs in the peripheral blood of juvenile patients with asthma. METHODS: In this study, we used hybridization arrays to compare the miRNA expression profiles among 6 juvenile patients with or without asthma. Using quantitative PCR (qPCR), we verified the expression levels of these miRNAs in plasma from patients with asthma (n = 40) and healthy subjects (n = 14). RESULTS: Our results showed that the levels of plasma miR-Let7C, miR-486, and miR-1260a in childhood asthma patients were significantly higher than in healthy controls (p < 0.01). Additionally, miR-1260a is correlated with the treatment schedule of these patients and patients with long treatment times had higher expression of miR-1260a than short treatment times; miR494 was significantly associated with challenge, and miR-3162-3p was significantly associated with MEF25 in asthma patients suggesting a potential correlation of miRNA levels with clinical disease parameters. Receiver operator characteristic analysis confirmed that the levels of miR-3162-3p could be used to discriminate childhood asthma patients from healthy subjects (area under the curve of 0.821), suggesting it may be a potential diagnostic biomarker. CONCLUSIONS: These results indicate that circulating miR-3162-3p and miR-1260a should be further evaluated as potential non-invasive biomarkers in diagnosis and treatment for childhood asthma.

Studies on microRNAs that are correlated with the cancer stem cells in chronic myeloid leukemia.

Accumulating data indicate that cancer stem cells play an important role in tumorigenesis and are underlying cause of tumor recurrence and metastasis, specifically in chronic myeloid leukemia (CML). We aim to detect the miRNAs that are correlated with the cancer stem cells in CML to provide theoretical basis for clinical application. We first analyzed microRNA expression profiles of CML leukemia patients compared with normal controls by microarray analysis and validated the results by real-time PCR. A single microRNA signature classified CML from normal was detected. We also determined the absolute copy numbers of these three microRNAs in normal adults. The results showed that three microRNAs (miR-150, miR-23a, and miR-130a) were identified to significantly decrease in expanded 38 CML patients compared with 90 normal controls. Molecular and statistical analysis showed that the decreased microRNAs were significant in clinical analysis. All these results indicated that those three microRNAs could act as a tumor suppressor and their decreased expression might be one of the causes of leukemia. Accordingly, clarifying their regulatory mechanisms might delineate their potentials as drug targets of gene therapy for CML.

Recapitulating and reversing human brain ribosomopathy defects via the maladaptive integrated stress response.

Animal or human models recapitulating brain ribosomopathies are incomplete, hampering development of urgently needed therapies. Here, we generated genetic mouse and human cerebral organoid models of brain ribosomopathies, caused by mutations in small nucleolar RNA (snoRNA) SNORD118. Both models exhibited protein synthesis loss, proteotoxic stress, and p53 activation and led to decreased proliferation and increased death of neural progenitor cells (NPCs), resulting in brain growth retardation, recapitulating features in human patients. Loss of SNORD118 function resulted in an aberrant upregulation of p-eIF2α, the mediator of integrated stress response (ISR). Using human iPSC cell-based screen, we identified small-molecule 2BAct, an ISR inhibitor, which potently reverses mutant NPC defects. Targeting ISR by 2BAct mitigated ribosomopathy defects in both cerebral organoid and mouse models. Thus, our SNORD118 mutant organoid and mice recapitulate human brain ribosomopathies and cross-validate maladaptive ISR as a key disease-driving mechanism, pointing to a therapeutic intervention strategy.