Establishment and Application of a Triplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assay for Differentiation of PEDV, TGEV and PKV.

The pathogens responsible for porcine viral diarrhea are diverse, causing significant economic losses to the pig industry. PEDV and TGEV are well-known pathogens causing diarrheal diseases in pigs, leading to significant economic losses in the breeding industry. In contrast, the newly identified diarrhea virus, PKV, has not garnered as much attention. However, co-infection of PKV with PEDV results in more severe symptoms in piglets, such as acute gastroenteritis, and promotes increased replication of PEDV. Rapid and accurate diagnosis of viral diarrhea is essential for farms to identify pathogens early and mitigate economic losses. This study describes the development of a triplex real-time fluorescent quantitative RT-qPCR technique that can simultaneously detect three RNA viruses associated with porcine viral diarrhea: PEDV, TGEV, and PKV. To establish the triplex RT-qPCR method for the simultaneous detection and identification of the above three diarrhea viruses, conserved regions of the M gene of TGEV, the N gene of PEDV, and the 3D gene of PKV were selected to design specific primers and probes. After optimizing the reaction conditions, the method's specificity, sensitivity, and reproducibility were evaluated. The triplex RT-qPCR method did not show a significant difference in PCR efficiency compared to the single RT-qPCR method. The method is specific to TGEV, PKV, and PEDV, exhibits no cross-reactivity with other pathogens, and demonstrates satisfactory sensitivity and reproducibility; the limit of detection (LOD) of PEDV, TGEV, and PKV is 11.42 copies/µL. Furthermore, the performance of the triplex RT-qPCR assay was compared with the Chinese standard single-assay method for detecting TGEV, PKV, and PEDV, showing complete consistency between the two methods (100% compliant). Subsequently, 1502 clinical diarrhea samples were collected from the Guangxi Zhuang Autonomous Region to investigate the local prevalence of TGEV, PKV, and PEDV and the positive rates were 16.38% (246/1502), 1.46% (22/1502), and 45.14% (678/1502), respectively. Co-infection of PEDV and PKV were most common, with a rate of 12.12% (182/1502). This study presents a valuable method for the rapid and simultaneous identification of PEDV, TGEV, and PKV in clinical animal farming practices, and provides a reassessment of the epidemiology of these diarrhea-causing viral pathogens in the Guangxi Zhuang Autonomous Region.

From deterministic to stochastic: an interpretable stochastic model-free reinforcement learning framework for portfolio optimization.

As a fundamental problem in algorithmic trading, portfolio optimization aims to maximize the cumulative return by continuously investing in various financial derivatives within a given time period. Recent years have witnessed the transformation from traditional machine learning trading algorithms to reinforcement learning algorithms due to their superior nature of sequential decision making. However, the exponential growth of the imperfect and noisy financial data that is supposedly leveraged by the deterministic strategy in reinforcement learning, makes it increasingly challenging for one to continuously obtain a profitable portfolio. Thus, in this work, we first reconstruct several deterministic and stochastic reinforcement algorithms as benchmarks. On this basis, we introduce a risk-aware reward function to balance the risk and return. Importantly, we propose a novel interpretable stochastic reinforcement learning framework which tailors a stochastic policy parameterized by Gaussian Mixtures and a distributional critic realized by quantiles for the problem of portfolio optimization. In our experiment, the proposed algorithm demonstrates its superior performance on U.S. market stocks with a 63.1% annual rate of return while at the same time reducing the market value max drawdown by 10% when back-testing during the stock market crash around March 2020.

ZNF300 promotes chemoresistance and aggressive behaviour in non-small-cell lung cancer.

OBJECTIVES: Chemoresistance induced by cisplatin has become the major impediment to lung cancer chemotherapy. This study explored the potential chemoresistant genes and underlying mechanisms of chemoresistance in NSCLC. MATERIALS AND METHODS: Gene expression profile was integrated with DNA methylation profile to screen the candidate chemoresistant genes. Bioinformatic analysis and immunohistochemistry were used to analyse the association of a candidate gene with the characteristics of NSCLC patients. Recombinant lentivirus vectors were utilized to overexpress or silence candidate gene. Microarrays and immunoblotting were applied to explore the downstream targets of candidate gene. Xenograft models were established to validate the findings in vitro. RESULTS: An increased ZNF300 expression was detected in three chemoresistant cell lines of NSCLC, and the higher expression of ZNF300 was associated with poor OS of NSCLC patients. Cells with upregulated ZNF300 presented chemoresistance and enhanced aggressive growth compared to cells with downregulated ZNF300. ZNF300 inhibited MAPK/ERK pathways and activated CDK1 through inhibiting WEE1 and MYT1 and modulating MYC/AURKA/BORA/PLK1 axis. ICA and ATRA improved the anti-tumour effect of cisplatin on chemoresistant cells by inducing differentiation. CONCLUSIONS: ZNF300 promotes chemoresistance and aggressive behaviour of NSCLC through regulation of proliferation and differentiation by downregulating MAPK/ERK pathways and regulation of slow-cycling phenotype via activating CDK1 by inhibiting WEE1/MYT1 and modulating MYC/AURKA/BORA/PLK1 axis. Cisplatin, combined with ATRA and ICA, might be beneficial in chemoresistant cases of NSCLC.

Tumor angiogenesis of SCLC inhibited by decreased expression of FMOD via downregulating angiogenic factors of endothelial cells.

Fibromodulin (FMOD), an ECM small leucine-rich proteoglycan (SLRP), was reported to promote angiogenesis not only during wound healing, but also in optical and cutaneous angiogenesis-dependent diseases. However, whether it plays important roles in tumor angiogenesis remains unclear. To explore the role of FMOD in tumor angiogenesis of human small cell lung cancer (SCLC), initially the study analyzed the relationship of FMOD expression in cancer tissues of SCLC with clinical characteristics. The analysis revealed that the positive FMOD expression was significantly associated with extensive stage of SCLC and higher vascular density. In mouse models, xenograft tumors developed with FMOD-silenced H446 cells (H446-shFMOD) exhibited slowed growth rate, decreased microvessel density, and reduced blood perfusion related to that of controls (H446-shCON). Additionally, compared with that of controls, the decreased secretion of FMOD in conditioned medium (CM) from H446-shFMOD inhibited proliferation, migration, and invasion of human umbilical vessel endothelial cells (HUVECs). Moreover, the decreased secretion of FMOD downregulated the expression of VEGF, TGF-ß1, FGF-2, and PDGF-B in HUVECs. The findings strongly suggested that the autocrine FMOD of cancer cells may promote tumor angiogenesis of SCLC by upregulating the expression of angiogenic factors that act in concert to facilitate the angiogenic phenotype of endothelial cells as a proangiogenic factor. Therefore, silencing FMOD may be a potentially clinical therapy for repressing tumor angiogenesis.

High-Performance Ratiometric Electrochemical Method Based on the Combination of Signal Probe and Inner Reference Probe in One Hairpin-Structured DNA.

In this work, the dual signal-tagged hairpin structured DNA (dhDNA)-based ratiometric probe was developed by the combination of ferrocene-labeled signal probe (Fc-sP) and methylene blue-modified inner reference probe (MB-rP) in one hairpin-structured DNA. On the basis of this, a high-performance ratiometric electrochemical method was proposed for biomarker detection. In contrast to the conventional ratiometric electrochemical probe, this dhDNA ratiometric probe integrated sP and rP into one structure, which ensured the completely same modification condition and the interdependence of sP and rP on one sensing interface. As a result, the dhDNA ratiometric probe possesses a stronger ability to eliminate the disturbance of environmental change, which was proven by the fact that the changes of the surface roughness and pH value had no significant effects on the reproducibility and stability of the sensor. Moreover, in the proposed strategy, the initial ratio responses of Fc-sP to MB-rP ((IFc-sP/IMB-rP)0) are controllable and can be kept constant at 1:1, which is favorable for the increase in signal-to-noise ratio and sensitivity. When the sequence of Fc-sP is designed as the aptamer of mucin 1 (MUC1), the dhDNA ratiometric sensor with signal amplification of Au nanoparticles becomes feasible for the sensitive detection of MUC1 by one-step incubation procedure. Compared with the conventional ratiometric sensor, the proposed dhDNA sensor has higher reproducibility, accuracy, stability, sensitivity, and simplicity, which are significant for the development of the sensor in various fields for practical applications.

The breast cancer susceptibility-related polymorphisms at the TOX3/LOC643714 locus associated with lung cancer risk in a Han Chinese population.

It has been well established that besides environmental factors, genetic factors are also associated with lung cancer risk. However, to date, the prior identified genetic variants and loci only explain a small fraction of the familial risk of lung cancer. Hence it is vital to investigate the remaining missing heritability to understand the development and process of lung cancer. In the study, to test our hypothesis that the previously identified breast cancer risk-associated genetic polymorphisms at the TOX3/LOC643714 locus might contribute to lung cancer risk, 16 SNPs at the TOX3/LOC643714 locus were evaluated in a Han Chinese population based on a case-control study. Pearson's chi-square test or Fisher's exact test revealed that rs9933638, rs12443621, and rs3104746 were significantly associated with lung cancer risk (P < 0.001, P < 0.001, and P = 0.005, respectively). Logistic regression analyses displayed that lung cancer risk of individuals with rs9933638(GG+GA) were 1.89 times higher than that of rs9933638AA carriers (OR = 1.893, 95% CI = 1.308-2.741, P = 0.001). Similar findings were manifested for rs12443621 (OR = 1.824, 95% CI = 1.272-2.616, P = 0.001, rs12443621(GG+GA) carriers vs. rs12443621AA carriers) and rs3104746 (OR = 1.665, 95% CI = 1.243-2.230, P = 0.001, rs3104746TT carriers vs. rs3104746(TA+AA) carriers). The study discovered for the first time that three SNPs (rs9933638, rs12443621, and rs3104746) at the TOX3/LOC643714 locus contributed to lung cancer risk, providing new evidences that lung cancer and breast cancer are linked at the molecular and genetic level to a certain extent.

Silencing of Paralemmin-3 Protects Mice from lipopolysaccharide-induced acute lung injury.

Excessive inflammatory response induced by lipopolysaccharide (LPS) plays a critical role in the development of acute lung injury (ALI). Paralemmin-3 (PALM3) is a novel protein that can modulate LPS-stimulated inflammatory responses in alveolar epithelial A549 cells. However, it remains unclear whether it is involved in the progression of ALI in vivo. Therefore, we studied the role of PALM3 in the pathogenesis of ALI induced by LPS. ALI was induced by LPS peritoneal injection in C57BL/6J mice. Lentivirus-mediated small interfering RNA (siRNA) targeting the mouse PALM3 gene and a negative control siRNA were intranasally administered to the mice. We found that the expression of PALM3 was up-regulated in the lung tissues obtained from the mouse model of LPS-induced ALI. The LPS-evoked inflammatory response (neutrophils and the concentrations of proinflammatory cytokines [IL-6, IL-1ß, TNF-α, MIP-2] in the bronchoalveolar lavage fluid [BALF]), histologic lung injury (lung injury score), permeability of the alveolar capillary barrier (lung wet/dry weight ratio and BALF protein concentration) and mortality rates were attenuated in the PALM3 siRNA-treated mice. These results indicate that PALM3 contributes to the development of ALI in mice challenged with LPS. Inhibiting PALM3 through the intranasal application of specific siRNA protected against LPS-induced ALI.

A novel and label-free biosensors for uracil-DNA glycosylase activity based on the electrochemical oxidation of guanine bases at the graphene modified electrode.

Uracil-DNA glycosylase (UDG) as an important base excision repair enzymes is widely distributed in organism, and it plays a crucial role in sustaining the genome integrity. Therefore, it is significant to carry out the analysis of UDG activity. In this present work, a novel and label-free electrochemical sensing platform for the sensitive detection of uracil DNA glycosylase (UDG) activity has been developed. Herein, the graphene modified glassy carbon (GC) electrode was prepared. And two complementary DNA strands were hybridized to form dsDNA (P1P2). In the presence of UDG, the uracil bases in P1P2 were specifically hydrolyzed, inducing the unwinding of the DNA duplex, and accompanied by the release of P1. Thus, the released P1 was adsorbed onto the graphene/GC electrode surface via π-π stacking. By investigating the electrochemical behavior of P1 at the graphene/GC electrode, the electrochemical oxidation of guanine bases in P1 was obviously observed. Therefore, using the current responses of guanine base in P1 as a signal indicator, UDG activity can be simply determined with high sensitivity, and the detectable lowest concentration is 0.01U/mL. This present design does not need covalent attachment of redox indicator to DNA, preventing participation of redox labels in the background. Meanwhile, the proposed strategy for the assay of UDG activity also has a remarkable sensitivity due to the excellent properties of graphene, which could increase both the immobilization amount of released ssDNA and the conductivity of the sensing system. All these elucidate that this developed protocol may lay a potential foundation for the sensitive detection of UDG activity in clinical diagnosis.

Lipopolysaccharide Induces Autophagic Cell Death through the PERK-Dependent Branch of the Unfolded Protein Response in Human Alveolar Epithelial A549 Cells.

BACKGROUND: Alveolar epithelial cell death plays a critical role in the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury. Increased autophagy has a dual effect on cell survival. However, it is not known whether autophagy promotes death or survival in human alveolar epithelial cells exposed to LPS. METHODS: Genetic and pharmacological approaches were used to evaluate the effect of autophagy on A549 cell viability upon LPS exposure. The endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways were examined with immunoblotting studies to further explore underlying mechanisms. RESULTS: Treatment with LPS (50 µg/ml) led to autophagy activation and decreased cell viability in A549 cells. Blocking autophagy via short interfering RNA or inhibitor significantly decreased, whereas rapamycin increased, the LPS-induced effect on viability. ER stress was activated in LPS-stimulated A549 cells, and ER stress inhibitor reduced LPS-induced autophagy. LPS activated only the PERK pathway and had rarely effect on the ATF6 and IRE1 branches of the UPR in A549 cells. Moreover, the knockdown of PERK and ATF4 attenuated LPS-induced autophagy and promoted cell survival. CONCLUSION: In human alveolar epithelial A549 cells, LPS induces autophagic cell death that depends on the activation of the PERK branch of the UPR upon ER stress.

Increased sensitivity of human lung adenocarcinoma cells to cisplatin associated with downregulated contactin-1.

Contactin-1 (CNTN-1), a glycosyl phosphatidylinositol anchor neural cell adhesion molecule (ACAM), is thought to function not only in nervous system development but also in the invasion and metastasis of several tumours. To investigate whether CNTN-1 is involved in multidrug resistance (MDR) in lung adenocarcinoma, CNTN-1 expression was compared between MDR human lung adenocarcinoma A549/cisplatin (A549/DDP) cells and its progenitor A549 cells. The comparison showed that CNTN-1 expression in A549/DDP cells was significantly higher than in A549 cells both at the mRNA level and the protein level. In order to confirm the physiological function of the abnormal expression, lentivirus-mediated short hairpin RNA (shRNA) was used to silence CNTN-1. Cell cytotoxicity assay and cell apoptosis assay revealed that silencing CNTN-1 both in A549 cells and in A549/DDP cells not only rendered cells more sensitive to cisplatin than the negative control, but also increased the cisplatin-induced apoptosis. Metastasis and invasion assays demonstrated that CNTN-1 knockdown reduced metastasis and invasion but did not affect A549 or A549/DDP cell proliferation. To investigate whether the abnormal expression of CNTN-1 is associated with characteristics of patients with non-small cell lung cancer (NSCLC), immunohistochemistry was used to detect CNTN-1 expression in 143 tissue samples from NSCLC patients and the results showed that the degree of CNTN-1 expression positively correlated with lymphatic invasion in patients with lung adenocarcinoma who received adjuvant cisplatin- or carboplatin-based treatment after surgery. Thus, we concluded that CNTN-1 is closely related with MDR of lung adenocarcinoma. Additionally, CNTN-1 is a novel marker to predict chemotherapeutic efficacy of patients with lung adenocarcinoma, especially with regard to cisplatin- or carboplatin-based regimens.

Silencing Angiopoietin-Like Protein 4 (ANGPTL4) Protects Against Lipopolysaccharide-Induced Acute Lung Injury Via Regulating SIRT1 /NF-kB Pathway.

Lung inflammation and alveolar epithelial cell death are critical events in the development and progression of acute lung injury (ALI). Although angiopoietin-like protein 4 (ANGPTL4) participates in inflammation, whether it plays important roles in ALI and alveolar epithelial cell inflammatory injury remains unclear. We therefore investigated the role of angptl4 in lipopolysaccharide (LPS)-induced ALI and the associated mechanisms. Lentivirus-mediated short interfering RNA targeted to the mouse angptl4 gene (AngsiRNA) and a negative control lentivirus (NCsiRNA) were intranasally administered to mice. Lung inflammatory injury and the underlying mechanisms for regulation of angptl4 on the LPS-induced ALI were subsequently determined. We reported that angptl4 levels were increased both in human alveolar epithelial A549 cells and lung tissues obtained from a mouse model of LPS-induced ALI. Angptl4 expression was induced by LPS in alveolar epithelial cells, whereas LPS-induced lung inflammation (neutrophils infiltration in the lung tissues, tumor necrosis factor α, interleukin 6), lung permeability (lung wet/dry weight ratio and bronchoalveolar lavage fluid (BALF) protein concentration), tissue damage (caspase3 activation), and mortality rates were attenuated in AngsiRNA-treated mice. The inflammatory reaction (tumor necrosis factor α, interleukin 6) and apoptosis rates were reduced in AngsiRNA(h)-treated A549 cells. Moreover, angptl4 promoted NF-kBp65 expression and suppressed SIRT1 expression both in mouse lungs and A549 cells. Additionally, SIRT1 antagonist nicotinamide (NAM) attenuated the inhibitory effects of AngsiRNA both on LPS-induced NF-kBp65 expression and IL6 expression. These findings suggest that silencing angptl4 protects against LPS-induced ALI via regulating SIRT1/NF-kB signaling pathway.

Inhibition of hypoxia-induced proliferation of pulmonary arterial smooth muscle cells by a mTOR siRNA-loaded cyclodextrin nanovector.

The proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key pathophysiological component of vascular remodeling in pulmonary arterial hypertension (PAH), an intractable disease, for which pharmacotherapy is limited and only slight improvement in survival outcomes have achieved over the past few decades. RNA interference provides a highly promising strategy to the treatment of this chronic lung disease, while efficient delivery of small interfering RNA (siRNA) remains a key challenge for the development of clinically acceptable siRNA therapeutics. With the aim to construct useful nanomedicines, the mammalian target of rapamycin (mTOR) siRNA was loaded into hybrid nanoparticles based on low molecular weight (Mw) polyethylenimine (PEI) and a pH-responsive cyclodextrin material (Ac-aCD) or poly(lactic-co-glycolic acid) (PLGA). This hybrid nanoplatform gave rise to desirable siRNA loading, and the payload release could be modulated by the hydrolysis characteristics of carrier materials. Fluorescence observation and flow cytometry quantification suggested that both Ac-aCD and PLGA nanovectors (NVs) may enter PASMCs under either normoxia or hypoxia conditions as well as in the presence of serum, with uptake and transfection efficiency significantly higher than those of cationic vectors such as PEI with Mw of 25 kDa (PEI25k) and Lipofectamine 2000 (Lipo 2k). Hybrid Ac-aCD or PLGA NV containing siRNA remarkably inhibited proliferation and activated apoptosis of hypoxic PASMCs, largely resulting from effective suppression of mTOR signaling as evidenced by significantly lowered expression of mTOR mRNA and phosphorylated protein. Moreover, these hybrid nanomedicines were more effective than commonly used cationic vectors like PEI25k and Lipo 2k, with respect to cell growth inhibition, apoptosis activation, and expression attenuation of mTOR mRNA and protein. Therefore, mTOR siRNA nanomedicines based on hybrid Ac-aCD or PLGA NV may be promising therapeutics for diseases related to hypoxic abnormal growth of PASMCs.

Promoter methylation profiles between human lung adenocarcinoma multidrug resistant A549/cisplatin (A549/DDP) cells and its progenitor A549 cells.

Although aberrant DNA methylation has been implicated in the pathophysiology of lung cancer, the role of methylation in multidrug resistance (MDR) of lung cancer has remained unclear. To investigate whether certain distinct DNA methylation pattern is associated with acquired MDR of lung adenocarcinoma, methylated-DNA immunoprecipitation-chromatin immunoprecipitation (MeDIP-ChIP) was utilised to compare the genome-wide promoter methylation of the human lung adenocarcinoma MDR A549/cisplatin (A549/DDP) cells with its progenitor A549 cells. The comparison identified 3617 genes with differentially methylated promoter, of which 1581 were hypermethylated and 2036 were hypomethylated. Then, bisulphite sequencing polymerase chain reaction (PCR) (BSP) and quantitative reverse transcription (RT)-PCR (Q-PCR) were used to validate the promoter methylation of five candidate genes and to determine whether the expression of genes was associated with the promoter methylation. BSP confirmed that the promoter methylation incidence of the hypermethylated genes, G protein-coupled receptor 56 isoform 3 (GPR56), metallothionein 1G (MT1G), and RAS association domain family gene 1 (RASSF1), was significantly higher in A549/DDP cells compared with A549 cells (p<0.001, p=0.0099, and p=0.0165), whereas no significant difference was found in that of the other two genes, CCNL2 and BAD (p=0.0594 and p=0.5546). Additionally, Q-PCR showed that the mRNA expression of the three hypermethylated genes was significantly lower in A549/DDP cells compared with A549 cells (all p<0.001). In conclusion, this study reported for the first time that a distinct promoter methylation pattern is associated with MDR of lung adenocarcinoma A549/DDP cells and suggested that GPR56, MT1G, and RASSF1 might be the potential methylation markers associated with acquired MDR of lung adenocarcinoma.

Regulatory effects of the JAK3/STAT1 pathway on the release of secreted phospholipase A2-IIA in microvascular endothelial cells of the injured brain.

BACKGROUND: Secreted phospholipase A2-IIA (sPLA2-IIA) is an inducible enzyme released under several inflammatory conditions. It has been shown that sPLA2-IIA is released from rat brain astrocytes after inflammatory stimulus, and lipopolysaccharide (LPS) and nitric oxide (NO) have been implicated in regulation of this release. Here, brain microvascular endothelial cells (BMVECs) were treated with LPS to uncover whether sPLA2-IIA was released, whether nitric oxide regulated this release, and any related signal mechanisms. METHODS: Supernatants were collected from primary cultures of BMVECs. The release of sPLA2-IIA, and the expression of inducible nitric oxide synthase (iNOS), phospho-JAK3, phospho-STAT1, total JAK3 and STAT1, ß-actin, and bovine serum albumin (BSA) were analyzed by Western blot or ELISA. NO production was calculated by the Griess reaction. sPLA2 enzyme activity was measured with a fluorometric assay. Specific inhibitors of NO (L-NAME and aminoguanidine, AG), JAK3 (WHI-P154,WHI), STAT1 (fludarabine, Flu), and STAT1 siRNA were used to determine the involvement of these molecules in the LPS-induced release of sPLA2-IIA from BMVECs. Nuclear STAT1 activation was tested with the EMSA method. The monolayer permeability of BMVECs was measured with a diffusion assay using biotinylated BSA. RESULTS: Treatment of BMVECs with LPS increased the release of sPLA2-IIA and nitrite into the cell culture medium up to 24 h. Pretreatment with an NO donor, sodium nitroprusside, decreased LPS-induced sPLA2-IIA release and sPLA2 enzyme activity, and enhanced the expression of iNOS and nitrite generation after LPS treatment. Pretreatment with L-NAME, AG, WHI-P154, or Flu notably reduced the expression of iNOS and nitrite, but increased sPLA2-IIA protein levels and sPLA2 enzyme activity. In addition, pretreatment of the cells with STAT1 siRNA inhibited the phosphorylation of STAT1, iNOS expression, and nitrite production, and enhanced the release of sPLA2-IIA. Pretreatment with the specific inhibitors of NOS, JAK2, and STAT3 decreased the permeability of BMVECs. In contrast, inhibition of sPLA2-IIA release increased cell permeability. These results suggest that sPLA2-IIA expression is regulated by the NO-JAK3-STAT1 pathway. Importantly, sPLA2-IIA augmentation could protect the LPS-induced permeability of BMVECs. CONCLUSION: Our results demonstrate the important action of sPLA2-IIA in the permeability of microvascular endothelial cells during brain inflammatory events. The sPLA2 and NO pathways can be potential targets for the management of brain MVEC injuries and related inflammation.

Mitochondrial DNA variant associated with Leber hereditary optic neuropathy and high-altitude Tibetans.

The distinction between mild pathogenic mtDNA mutations and population polymorphisms can be ambiguous because both are homoplasmic, alter conserved functions, and correlate with disease. One possible explanation for this ambiguity is that the same variant may have different consequences in different contexts. The NADH dehydrogenase subunit 1 (ND1) nucleotide 3394 T > C (Y30H) variant is such a case. This variant has been associated with Leber hereditary optic neuropathy and it reduces complex I activity and cellular respiration between 7% and 28% on the Asian B4c and F1 haplogroup backgrounds. However, complex I activity between B4c and F1 mtDNAs, which harbor the common 3394T allele, can also differ by 30%. In Asia, the 3394C variant is most commonly associated with the M9 haplogroup, which is rare at low elevations but increases in frequency with elevation to an average of 25% of the Tibetan mtDNAs (odds ratio = 23.7). In high-altitude Tibetan and Indian populations, the 3394C variant occurs on five different macrohaplogroup M haplogroup backgrounds and is enriched on the M9 background in Tibet and the C4a4 background on the Indian Deccan Plateau (odds ratio = 21.9). When present on the M9 background, the 3394C variant is associated with a complex I activity that is equal to or higher than that of the 3394T variant on the B4c and F1 backgrounds. Hence, the 3394C variant can either be deleterious or beneficial depending on its haplogroup and environmental context. Thus, this mtDNA variant fulfills the criteria for a common variant that predisposes to a "complex" disease.

Association of mitochondrial DNA variations with lung cancer risk in a Han Chinese population from southwestern China.

Mitochondrial DNA (mtDNA) is particularly susceptible to oxidative damage and mutation due to the high rate of reactive oxygen species (ROS) production and limited DNA-repair capacity in mitochondrial. Previous studies demonstrated that the increased mtDNA copy number for compensation for damage, which was associated with cigarette smoking, has been found to be associated with lung cancer risk among heavy smokers. Given that the common and "non-pathological" mtDNA variations determine differences in oxidative phosphorylation performance and ROS production, an important determinant of lung cancer risk, we hypothesize that the mtDNA variations may play roles in lung cancer risk. To test this hypothesis, we conducted a case-control study to compare the frequencies of mtDNA haplogroups and an 822 bp mtDNA deletion between 422 lung cancer patients and 504 controls. Multivariate logistic regression analysis revealed that haplogroups D and F were related to individual lung cancer resistance (OR = 0.465, 95%CI = 0.329-0.656, p<0.001; and OR = 0.622, 95%CI = 0.425-0.909, p = 0.014, respectively), while haplogroups G and M7 might be risk factors for lung cancer (OR = 3.924, 95%CI = 1.757-6.689, p<0.001; and OR = 2.037, 95%CI = 1.253-3.312, p = 0.004, respectively). Additionally, multivariate logistic regression analysis revealed that cigarette smoking was a risk factor for the 822 bp mtDNA deletion. Furthermore, the increased frequencies of the mtDNA deletion in male cigarette smoking subjects of combined cases and controls with haplogroup D indicated that the haplogroup D might be susceptible to DNA damage from external ROS caused by heavy cigarette smoking.

Restoration of SOCS3 suppresses human lung adenocarcinoma cell growth by downregulating activation of Erk1/2, Akt apart from STAT3.

SOCS3 is regarded as a major negative regulator of STAT3. Recent evidence indicates that SOCS3 regulates strength and duration of other signaling pathways including ras/ERK1/2/MAPK, PI3-K/Akt in non-malignant cells. The repression or silence of SOCS3 expression in a few tumor types has led to speculation that loss of SOCS3 gene is closely related to deregulation of multiple signal pathways during tumorigenesis. However, apart from STAT3, little is known in malignant cells about the mechanism by which SOCS3 modulates other intracellular signal cascades such as Erk1/2 and Akt, whose aberrant activation has been implicated in many human tumors. Expression of SOCS3 proved deficient in human lung adenocarcinoma A549 cells, and forced expression of SOCS3 resulted in growth inhibition. Growth suppression due to SOCS3 was associated with attenuated activation of Erk1/2, Akt as well as STAT3. The results suggested that SOCS3, as negative regulators of cytokine signaling, might maintain homeostasis by regulating multiple signaling pathways and reverse cell malignant behavior.

Sequential activation of JAKs, STATs and xanthine dehydrogenase/oxidase by hypoxia in lung microvascular endothelial cells.

Xanthine dehydrogenase/oxidase (XDH/XO) is associated with various pathological conditions related to the endothelial injury. However, the molecular mechanism underlying the activation of XDH/XO by hypoxia remains largely unknown. In this report, we determined whether the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) signaling pathway is involved in hypoxia-induced activation of XDH/XO in primary cultures of lung microvascular endothelial cells (LMVEC). We found that hypoxia significantly increased interleukin 6 (IL6) production in a time-dependent manner in LMVEC. Hypoxia also markedly augmented phosphorylation/activation of JAKs (JAK1, JAK2 and JAK3) and the JAK downstream effectors STATs (STAT3 and STAT5). Hypoxia-induced activation of STAT3 was blocked by IL6 antibodies, the JAK inhibitor AG490 and the suppressor of cytokine signaling 3 (SOCS3), implying that hypoxia-promoted IL6 secretion activates the JAK/STAT pathway in LMVEC. Phosphorylation and DNA-binding activity of STAT3 were also inhibited by the p38 MAPK inhibitor SB203580 and the phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that multiple signaling pathways involved in STAT activation by hypoxia. Importantly, hypoxia promoted XDH/XO activation in LMVEC, which was markedly reversed by inhibiting the JAK-STAT pathway using IL6 antibodies, AG490 and SOCS3. These data demonstrated that JAKs, STATs and XDH/XO were sequentially activated by hypoxia. These data provide the first evidence indicating that the JAK-STAT pathway is involved in hypoxia-mediated XDH/XO activation in LMVEC.

[Involvement of bcl-2 in multidrug resistance in human small cell lung cancer cell subline H446/DDP].

OBJECTIVE: To study the mechanism of bcl-2 involvement in multidrug resistance in human small cell lung cancer (SCLC) cell subline H446/DDP. METHODS: After the construction of pLXSN-bcl-2, in which the full length of bcl-2 cDNA amplified from total RNA of H446/DDP cells was integrated into the mammalian expression vector pLXSN, allowing transcription of a bicistronic mRNA, and the synthesis in vitro of 20-mer ODNs targeting the coding region of bcl-2 mRNA and the scrambled ODNs used as the control, the cationic lipid DOTAP was used to transfect them into H446 and H446/DDP cells, respectively. When H446 cells were transfected with mammalian expression vector pLXSN, the cells were divided into 3 groups, including cells transfected with the recombinant pLXSN-bcl-2, cells transfected with the vector pLXSN and cells untransfected (control); when H446 cells and H446/DDP cells were transfected with PS-ODNs, the cells were divided into 3 groups, including cells transfected with AS-PS-ODNs, cells transfected with NS-PS-ODNs and cells untransfected (control), respectively. After transfection, Western blot were carried out to detect the expression level of bcl-2 in the control cells and the transfected cells, respectively. Meanwhile flow cytometry (FCM) was used to detect cell apoptosis in the control cells and the transfected cells. RESULTS: (1) The data from Western blot showed that compared with the control H446 cells (gray-scale value 0.103 +/- 0.005), the expression level of bcl-2 in the pLXSN-bcl-2 transfected H446 cells (gray-scale value 0.854 +/- 0.016) was increased significantly (P < 0.01); and the apoptosis from DNA content analysis decreased significantly in the pLXSN-bcl-2 transfected H446 cells [(20.9 +/- 0.2)%] compared with that of the control H446 cells [(31.1 +/- 0.2)%] by DNA content analysis (P < 0.01). (2) The results from Western blot showed that bcl-2 expression in the AS-PS-ODNs transfected H446/DDP cells (gray-scale value 0.695 +/- 0.014) was significantly reduced compared with that of the control H446/DDP cells (gray-scale value 0.942 +/- 0.018), although not totally reduced to the level of the control H446 cells (P < 0.01); and the data from DNA content analysis indicated that the sensitivity of the AS-PS-ODNs transfected H446/DDP cells to 5 microg/ml DDP-induced apoptosis was strongly increased as compared with that of the control H446/DDP cells (P < 0.01). CONCLUSION: Targeting to inhibit antiapoptotic mitochondrial gene Bcl-2 expression may be one of the important therapeutic approaches to overcoming chemoresistance in human SCLC.

Enhancement of antiangiogenic effects of human canstatin with a hypoxia-regulated transgene vector in lung cancer model.

UNLABELLED: Canstatin, a newly identified antiangiogenesis protein, has a potent inhibitory effect on the proliferation and growth of endothelial cells. To enhance the expression and antiangiogenic effects of canstatin in solid tumors, we constructed a eukaryotic expression vector that encodes human canstatin cDNA downstream from nine copies of the hypoxia-response element. METHODS: Canstatin complementary DNA from adult liver tissues was cloned into the mammalian expression vector pCMV-Script. Nine copies of the hypoxia-response element were ligated upstream from the canstatin gene near the cytomegalovirus promoter. The recombinant vector, pCMV9Cans, was transformed into A549 cells by cationic liposomes. The transformed cells were cultured under oxic and anoxic conditions. We detected canstatin messenger ribonucleic acid and protein expression in transformed cells by TaqMan polymerase chain reaction and Western blot analysis, respectively. Human umbilical vein endothelial cells were cocultured with recombinant vector transformed A549 cells using Transwell plates under oxic and anoxic conditions. The proliferation and apoptosis of the cocultured endothelial cells were evaluated with 3H-thymidine incorporation and terminal deoxynucleotidyl-mediated biotinylated deoxyuridine triphosphate nick end-labeling methods (TUNEL), respectively. A canstatin-encoding vector with no hypoxia-response element, pCMVCans, was used as the positive control, and naked plasmid-transformed and singly cultured parental cells were used as negative controls. The biologic activity of the vector in tumor tissues of lung cancer-bearing nude mice was evaluated by microvessel counts. Canstatin protein expression was assessed by Western blot analysis in tumor tissues. pCMVCans and empty vector were used as controls in the in vivo assays. RESULTS: Canstatin messenger RNA and protein were detected in both pCMV9Cans- and pCMVCans-transformed A549 clones. Under oxic conditions, canstatin expression was not significantly different in clones stably transformed with pCMV9Cans or pCMVCans. However, under anoxic conditions canstatin expression was significantly higher in pCMV9Cans-transformed cells than in pCMVCans-transformed cells. Moreover, the 3H-thymidine uptake rate of the human umbilical vein endothelial cells was markedly lower than that of the pCMVCans-transformed cells, and many endothelial cells underwent apoptosis when cocultured with pCMV9Cans-transformed A549 cells, especially under anoxic conditions. We detected canstatin expression in tumor tissues; the expression level in pCMV9Cans-transformed tumors was significantly higher than that in pCMVCans-transformed tumors. An in vivo assay showed that tumors transformed with pCMV9Cans remained small, and microvessels in those tumors were much fewer than those in pCMVCans-transformed tumors. CONCLUSION: The hypoxia-regulated vector pCMV9Cans increases the expression of canstatin, thereby enhancing its biological effects. We believe that the hypoxia-inducible canstatin-expressing vector is a promising gene therapy tool for antiangiogenesis research.