A common whole-genome paleotetraploidization in Cucurbitales.

Cucurbitales are an important order of flowering plants known for encompassing edible plants of economic and medicinal value and numerous ornamental plants of horticultural value. By reanalyzing the genomes of two representative families (Cucurbitaceae and Begoniaceae) in Cucurbitales, we found that the previously identified Cucurbitaceae common paleotetraploidization that occurred shortly after the core-eudicot-common hexaploidization event is shared by Cucurbitales, including Begoniaceae. We built a multigenome alignment framework for Cucurbitales by identifying orthologs and paralogs and systematically redating key evolutionary events in Cucurbitales. Notably, characterizing the gene retention levels and genomic fractionation patterns between subgenomes generated from different polyploidizations in Cucurbitales suggested the autopolyploid nature of the Begoniaceae common tetraploidization and the allopolyploid nature of the Cucurbitales common tetraploidization and the Cucurbita-specific tetraploidization. Moreover, we constructed the ancestral Cucurbitales karyotype comprising 17 proto-chromosomes, confirming that the most recent common ancestor of Cucurbitaceae contained 15 proto-chromosomes and rejecting the previous hypothesis for an ancestral Cucurbitaceae karyotype with 12 proto-chromosomes. In addition, we found that the polyploidization and tandem duplication events promoted the expansion of gene families involved in the cucurbitacin biosynthesis pathway; however, gene loss and chromosomal rearrangements likely limited the expansion of these gene families.

Simulated wrapped phase optimizes phase retrieval in phase-shifting interferometry.

Phase retrieval is crucial in phase-shifting interferometry and other phase measurement techniques. However, in noisy wrapped phase maps with high steepness, discontinuities arise and cause phase unwrapping errors. To solve this problem, this Letter presents a phase retrieval method based on a simulated wrapped phase. By establishing the correspondence between the simulated and measured interferograms, the difference in wrapped phases between them can be obtained. The difference in wrapped phase map, which has sparse and wide interference fringes, has a higher reliability of phase unwrapping. The proposed method not only possesses high phase retrieval accuracy but it also simplifies the processing of interferograms. Furthermore, the layout of all interferometric systems, the parameters of optical components, and the model of the measured object are known, so the proposed method can be used as a reference for phase retrieval.

Investigation of the Sheltering Effects on the Mobilization of Microplastics in Open-Channel Flow.

Microplastics (MPs) can be sheltered by bed grains, resulting in a higher threshold of mobilization compared with those moving on a smooth bed. Only one formula considering the sheltering effects has been proposed for the critical shear stress (τc) of MP mobilization by including the densities and sizes of MPs and bed grains, but it is inaccurate for MPs of certain materials such as PS and shows limitations in understanding the sheltering effects. Additionally, no method exists for the critical depth-averaged velocity (Uc). In this study, experiments were conducted measuring both τc and Uc of MPs (made of PA, PVC, and PET) deposited on rough beds of different roughnesses, with a focus on the sheltering effects. A power law relationship between τc and the density and size of MPs was found, and a new formula for estimating τc was proposed for MPs, which reduced the errors by approximately 40% compared with that of the previous formula (27.8%). To explicitly quantify the sheltering effects in MP mobilizations, hiddenness (ΔZ), exposure (ΔH), and longitudinal exposure (ΔX) were introduced, all of which were observed to follow a normal distribution. A semiempirical method for Uc was then proposed incorporating the new measures, which reduced the errors by around 70% compared to the methods developed for estimating the Uc values of sediments.

Incipient Motion of Exposed Microplastics in an Open-Channel Flow.

The incipient motion threshold of microplastics (MPs), defined as the condition that is just sufficient to initiate MP movement, is key to assessing the transport and fate of MPs in water bodies, yet only a few studies have focused on its prediction. This study experimentally investigated the effects of bed roughness (smooth and rough beds) and MP properties (shapes, sizes, and densities) on the incipient velocity (Ui) and critical shear stress (τc) of exposed MPs in an open-channel flow. For a total of 19 types of MPs, Ui and τc were found to range from 0.06 to 0.21 m/s and 0.01 to 0.075 N/m2, respectively. The commonly used thresholds for sediment transport, for example, critical shield parameter Θc and movability number Λc, were established for MPs based on τc. Based on the experimental data of the literature and this study, it was found that predictions of Θc and Λc for sediments do not apply to MPs. A new explicit formula for Λc was proposed for predicting the incipient motion of MPs by introducing the dimensionless particle diameter (d*) and a new dimensionless parameter related to the particle size, density, and shape. The new formula has an absolute error of 12.3%, which is smaller than the existing formula for MPs (55.6%).

Deep Learning Approach for Detection of Underground Natural Gas Micro-Leakage Using Infrared Thermal Images.

The leakage of underground natural gas has a negative impact on the environment and safety. Trace amounts of gas leak concentration cannot reach the threshold for direct detection. The low concentration of natural gas can cause changes in surface vegetation, so remote sensing can be used to detect micro-leakage indirectly. This study used infrared thermal imaging combined with deep learning methods to detect natural gas micro-leakage areas and revealed the different canopy temperature characteristics of four vegetation varieties (grass, soybean, corn and wheat) under natural gas stress from 2017 to 2019. The correlation analysis between natural gas concentration and canopy temperature showed that the canopy temperature of vegetation increased under gas stress. A GoogLeNet model with Bilinear pooling (GLNB) was proposed for the classification of different vegetation varieties under natural gas micro-leakage stress. Further, transfer learning is used to improve the model training process and classification efficiency. The proposed methods achieved 95.33% average accuracy, 95.02% average recall and 95.52% average specificity of stress classification for four vegetation varieties. Finally, based on Grad-Cam and the quasi-circular spatial distribution rules of gas stressed areas, the range of natural gas micro-leakage stress areas under different vegetation and stress durations was detected. Taken together, this study demonstrated the potential of using thermal infrared imaging and deep learning in identifying gas-stressed vegetation, which was of great value for detecting the location of natural gas micro-leakage.

Stabilization of 4E-BP1 by PI3K kinase and its involvement in CHK2 phosphorylation in the cellular response to radiation.

Objectives: 4E-BP1 is a family member of eIF4E binding proteins (4E-BPs) which act as the suppressors of cap-dependent translation of RNA via competitively associating with cap-bound eIF4E. RNA translation regulation is an important manner to control the cellular responses to a series of stress conditions such as ionizing radiation (IR)-induced DNA damage response and cell cycle controlling. This study aimed to determine the mechanism of 4E-BP1 stabilization and its potential downstream target(s) in the response to IR. Methods: PI3Ks kinase inhibitors were used to determine the signaling control of 4E-BP1 phosphorylation and protein stability. shRNA strategy was employed to silence the expression of 4E-BP1 in HeLa and HepG2 cells, and determine its effect on the irradiation-induced CHK2 phosphorylation. The protein degradation/stability was investigated by western blotting on the condition of blocking novel protein synthesis by cycloheximide (CHX). Results: The phosphorylation of 4E-BP1 at Thr37/46 was significantly increased in both HepG2 and HeLa cells by ionizing radiation. Depression of 4E-BP1 by shRNA strategy resulted in an incomplete G2 arrest at the early stage of 2 hours post-irradiation, as well as a higher accumulation of mitotic cells at 10 and 12 hours post-irradiation as compared to the control cells. Consistently, the CHK2 phosphorylation at Thr68 induced by IR was also attenuated by silencing 4E-BP1 expression. Both PI3K and DNA-PKcs kinase inhibitors significantly decreased the protein level of 4E-BP1, which was associated with the accelerated degradation mediated by ubiquitination-proteasome pathway. Conclusion: PI3K kinase activity is necessary for maintaining 4E-BP1 stability. Our results also suggest 4E-BP1 a novel biological role of regulating cell cycle G2 checkpoint in responding to IR stress in association with controlling CHK2 phosphorylation.

Machine learning-based prediction for settling velocity of microplastics with various shapes.

Microplastics can easily enter the aquatic environment and be transported between water bodies. The terminal settling velocity of microplastics, which affects their transport and distribution in the aquatic environment, is mainly influenced by their size, density, and shape. Due to the difficulty in accurately predicting the terminal settling velocity of microplastics with various shapes, this study focuses on establishing high-performance prediction models and understanding the importance and effect of each feature parameter using machine learning. Based on the number of principal dimensions, the shapes of microplastics are classified into fiber, film, and fragment, and their thresholds are identified. The microplastics of different shape categories have different optimal shape parameters for predicting the terminal settling velocity: Corey shape factor, flatness, elongation, and sphericity for the fragment, film, fiber, and mixed-shape MPs, respectively. By including the dimensionless diameter, relative density and optimal shape parameter in the input parameter combination, the machine learning models can well predict the terminal settling velocity for the microplastics of different shape categories and mixed-shape with R2 > 0.867, achieving significantly higher performance than the existing theoretical and regression models. The interpretable analysis of machine learning reveals the highest importance of the microplastic size and its marginal effect when the dimensionless diameter D* = dn(g/v2)1/3 > 80, where dn is the equivalent diameter, g is the gravitational acceleration, and ν is the fluid kinematic viscosity. The effect of shape is weak for small microplastics and becomes significant when D* exceeds 65.

Interfaces Engineering of Ultrafine Ni@Ni2P/C Core-Shell Heterostructure for High Yield Hydrogen Peroxide Electrosynthesis.

Developing cost-effective and sustainable catalysts with exceptional activity and selectivity is essential for the practical implementation of on-site H2O2 electrosynthesis, yet it remains a formidable challenge. Metal phosphide core-shell heterostructures anchored in carbon nanosheets (denoted as Ni@Ni2P/C NSs) are designed and synthesized via carbonization and phosphidation of the 2D Ni-BDC precursor. This core-shell nanostructure provides more accessible active sites and enhanced durability, while the 2D carbon nanosheet substrate prevents heterostructure aggregation and facilitates mass transfer. Theoretical calculations further reveal that the Ni/Ni2P heterostructure-induced optimization of geometric and electronic structures enables the favored adsorption of OOH* intermediate. All these features endow the Ni@Ni2P/C NSs with remarkable performance in 2e ORR for H2O2 synthesis, achieving a top yield rate of 95.6 mg L-1 h-1 with both selectivity and Faradaic efficiency exceeding 90% under a wide range of applied potentials. Furthermore, when utilized as the anode of an assembled gas diffusion electrode (GDE) device, the Ni@Ni2P/C NSs achieve in situ H2O2 production with excellent long-term durability (>32 h). Evidently, this work provides a unique insight into the origin of 2e ORR and proposes optimization of H2O2 production through nano-interface manipulation.

Settling behaviors of microplastic disks in acceleration fall.

The settling of microplastics (MPs) in the initial acceleration fall stage, i.e., before reaching the terminal settling velocity, has not been investigated, which is however important for understanding MP transport and fate. MP disks sized 3-5 mm, of three shapes and made of three polymers (1.038-1.343 g/cm3) were examined. Five release ways and three release angles (0°, 45°, 90°) were used. MP disks with the release angle of 0° start to zigzag immediately after the release, while the MP disks with the release angles of 45° and 90° first adjust to a horizontal position and then zigzag. The adjustment distances in the vertical and horizontal directions, as well as the maximum vertical settling velocity, are influenced by MP density, size, release angle and release way. The detailed settling trajectory and velocity were also analyzed. Finally, the time-changing drag coefficient of MP disks was examined and discussed.

Settling behaviors of microplastic disks in water.

Microplastic (MP) disks have not been studied for settling behaviors in aquatic environments, which affects the transport and fate of MPs. Therefore, settling experiments were conducted on MP disks of three shapes and four common-seen materials. Lighter MP disks (with density ρs = 1.038 g/cm3 and length l ≤ 5 mm) followed rectilinear vertical trajectories, while heavier MP disks (ρs = 1.161-1.343 g/cm3 and l = 5 mm) followed zigzag trajectories with oscillations and rotations. The mean terminal settling velocities of MP disks were 19.6-48.8 mm/s. Instantaneous settling velocities of heavier MP disks fluctuated. Existing formulas could not accurately predict the settling velocity of MP disks; thus, a new model was proposed with an error of 15.5 %. Finally, the Red - I* diagram (Red is the disk Reynolds number and I* is the dimensionless moment of inertia) was extended for MP disks to predict settling trajectories.

Systemic Inflammation Response Index Predicts Clinical Outcomes in Patients With Acute Ischemic Stroke (AIS) After the Treatment of Intravenous Thrombolysis.

BACKGROUND: Intravenous thrombolysis (IVT) is one of the most important means of therapy for patients with acute ischemic stroke (AIS). After cerebral infarction, the inflammatory response fulfills an essential role in the pathobiology of stroke, affecting the process of recanalization. Hence, we evaluated the usefulness of the systemic inflammatory response index (SIRI) for the prognosis of patients with AIS. METHODS: A total of 161 patients suffering from AIS were retrospectively analyzed. SIRI was introduced and calculated using the absolute neutrophil, monocyte, and lymphocyte numbers from the admission blood work. The study outcomes were determined using a modified Rankin Scale (mRS) at the 3-month timepoint, and a favorable clinical outcome was calculated in the mRS score range of 0 to 2. The analysis of receiver operating characteristic (ROC) curves was performed to determine the values of the optimal cutoff of SIRI for the prediction of clinical outcomes. In addition, multivariate analyses were performed to investigate the association between clinical outcomes and SIRI. RESULTS: The ROC curve analysis revealed that the ideal SIRI cutoff was at 2.54 [area under the curve, 78.85%; 95% CI, 71.70% to 86.00%; sensitivity, 70.89%; and specificity, 84.14%]. Multivariate analysis indicated that SIRI ≤2.54 (odds ratio, 1.557, 95% CI, 1.269 to 1.840; P =0.021) was an independent predictor of favorable clinical outcomes in patients suffering from AIS after treatment with IVT. CONCLUSIONS: We preliminary speculate that SIRI may serve as an independent predictor of clinical outcomes with AIS following IVT.

Two-step model of paleohexaploidy, ancestral genome reshuffling and plasticity of heat shock response in Asteraceae.

An ancient hexaploidization event in the most but not all Asteraceae plants, may have been responsible for shaping the genomes of many horticultural, ornamental, and medicinal plants that promoting the prosperity of the largest angiosperm family on the earth. However, the duplication process of this hexaploidy, as well as the genomic and phenotypic diversity of extant Asteraceae plants caused by paleogenome reorganization, are still poorly understood. We analyzed 11 genomes from 10 genera in Asteraceae, and redated the Asteraceae common hexaploidization (ACH) event ~70.7-78.6 million years ago (Mya) and the Asteroideae specific tetraploidization (AST) event ~41.6-46.2 Mya. Moreover, we identified the genomic homologies generated from the ACH, AST and speciation events, and constructed a multiple genome alignment framework for Asteraceae. Subsequently, we revealed biased fractionations between the paleopolyploidization produced subgenomes, suggesting the ACH and AST both are allopolyplodization events. Interestingly, the paleochromosome reshuffling traces provided clear evidence for the two-step duplications of ACH event in Asteraceae. Furthermore, we reconstructed ancestral Asteraceae karyotype (AAK) that has 9 paleochromosomes, and revealed a highly flexible reshuffling of Asteraceae paleogenome. Of specific significance, we explored the genetic diversity of Heat Shock Transcription Factors (Hsfs) associated with recursive whole-genome polyploidizations, gene duplications, and paleogenome reshuffling, and revealed that the expansion of Hsfs gene families enable heat shock plasticity during the genome evolution of Asteraceae. Our study provides insights on polyploidy and paleogenome remodeling for the successful establishment of Asteraceae, and is helpful for further communication and exploration of the diversification of plant families and phenotypes.

A new model for the terminal settling velocity of microplastics.

Microplastic (MP) settling process is important for the transport of microplastic particles (MPs, <5 mm) in water bodies. However, for the control parameter of the drag coefficient (Cd), no generalized formula has been proposed for MPs of different shapes and materials. In this study, a total of 1343 MP settling data were collected from the literature. It was found that the drag law for perfect spheres cannot reasonably predict Cd for MPs with particle Reynolds number of 1-103. A new formula for Cd was developed by introducing the dimensionless particle diameter (d⁎) and two shape descriptors. The absolute error of the new formula is 15.2%, smaller than those (42.5-72.8%) of other existing formulas. Moreover, an explicit model was developed for MP settling velocity by correlating Cd, d⁎, and shape descriptors, with lower absolute error (8.8%) than those (15.4-77.2%) of existing models.

A Novel Clinically Prognostic Stratification Based on Prognostic Nutritional Index Status and Histological Grade in Patients With Gallbladder Cancer After Radical Surgery.

Purpose: Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract, with a 5-year survival rate of 5%. The prognostic models to predict the prognosis of patients with GBC remain controversial. Therefore, to construct a prognosis prediction of GBC, a retrospective cohort study was carried out to investigate the prognostic nutritional index and histological grade in the long-term outcome of patients with GBC after radical surgery (RS). Methods: A retrospective study of a total of 198 patients with GBC who underwent surgical treatment were enrolled. The hematological indicators, imageological data, and perioperative clinical data were acquired for statistical analysis and poor prognosis model construction. Results: Prognostic nutrition index (PNI) < 45.88, maximum tumor diameter (MTD) > 2.24 cm, and jaundice (JD) were all associated with a poor prognosis in multivariate logistic regression analysis. The prognosis prediction model was based on the three risk factors, which indicated a superior predictive ability in the primary cohort [area under the curve (AUC) = 0.951] and validation cohort (AUC = 0.888). In multivariate Cox regression analysis, poorly differentiation (PD) was associated with poor 3-year survival. In addition, Kaplan-Meier (KM) survival analysis suggested that GBC patients with high-risk scores and PD had a better prognosis after RS (p < 0.05), but there was no significant difference in prognosis for patients with non-poorly differentiation (NPD) or low-risk scores after RS (p > 0.05). Conclusion: Our prediction model for GBC patients with prognosis evaluation is accurate and effective. For patients with PD and high-risk scores, RS is highly recommended; a simple cholecystectomy can also be considered for acceptance for patients with NPD or low-risk score. The significant findings provide a new therapeutic strategy for the clinical treatment of GBC.

Helicobacter pylori and Alzheimer's Disease-Related Metabolic Dysfunction: Activation of TLR4/Myd88 Inflammation Pathway from p53 Perspective and a Case Study of Low-Dose Radiation Intervention.

Gut dysbiosis is observed in Alzheimer's disease (AD) and is frequently associated with AD-induced metabolic dysfunction. However, the extent and specific underlying molecular mechanisms triggered by alterations of gut microbiota composition and function mediating AD-induced metabolic dysfunction in AD remain incompletely uncovered. Here, we indicate that Helicobacter pylori (H. pylori) is abundant in AD patients with relative metabolic dysfunction. Fecal microbiota transplantation from the AD patients promoted metabolic dysfunction in mice and increased gut permeability. H. pylori increased gut permeability through activation of the TLR4/Myd88 inflammation pathway in a p53-dependent manner, leading to metabolic dysfunction. Moreover, p53 deficiency reduced bile acid concentration, leading to an increased abundance of H. pylori colonization. Overall, these data identify H. pylori as a key promoter of AD-induced metabolic dysfunction.

Two independent allohexaploidizations and genomic fractionation in Solanales.

Solanales, an order of flowering plants, contains the most economically important vegetables among all plant orders. To date, many Solanales genomes have been sequenced. However, the evolutionary processes of polyploidization events in Solanales and the impact of polyploidy on species diversity remain poorly understood. We compared two representative Solanales genomes (Solanum lycopersicum L. and Ipomoea triloba L.) and the Vitis vinifera L. genome and confirmed two independent polyploidization events. Solanaceae common hexaploidization (SCH) and Convolvulaceae common hexaploidization (CCH) occurred ∼43-49 and ∼40-46 million years ago (Mya), respectively. Moreover, we identified homologous genes related to polyploidization and speciation and constructed multiple genomic alignments with V. vinifera genome, providing a genomic homology framework for future Solanales research. Notably, the three polyploidization-produced subgenomes in both S. lycopersicum and I. triloba showed significant genomic fractionation bias, suggesting the allohexaploid nature of the SCH and CCH events. However, we found that the higher genomic fractionation bias of polyploidization-produced subgenomes in Solanaceae was likely responsible for their more abundant species diversity than that in Convolvulaceae. Furthermore, through genomic fractionation and chromosomal structural variation comparisons, we revealed the allohexaploid natures of SCH and CCH, both of which were formed by two-step duplications. In addition, we found that the second step of two paleohexaploidization events promoted the expansion and diversity of ß-amylase (BMY) genes in Solanales. These current efforts provide a solid foundation for future genomic and functional exploration of Solanales.

A novel miR-0308-3p revealed by miRNA-seq of HBV-positive hepatocellular carcinoma suppresses cell proliferation and promotes G1/S arrest by targeting double CDK6/Cyclin D1 genes.

BACKGROUND: Persistent infection with hepatitis B virus (HBV) accounts for the majority of hepatocellular carcinoma (HCC), but the molecular mechanisms underlying liver carcinogenesis are still not completely understood. Increasing evidence demonstrates that microRNAs (miRNAs) play significant functional roles in virus-host interactions. The aim of this study was to explore differentially expressed miRNA profiles and investigate the molecular mechanism of miR-0308-3p in HBV-positive HCC carcinogenesis. METHODS: High-throughput sequencing was used to detect novel miRNAs in three samples of HBV-positive HCC tissue compared to matched HBV-negative HCC tissue. The Cancer Genome Atlas (TCGA) database was used to mine miRNAs related to HBV-positive HCC. Bioinformatics analyses were conducted to predict the miRNAs' possible biological and pathway regulatory functions. Quantitative polymerase chain reaction (qPCR) was then applied to evaluate the expression levels of randomly selected miRNAs. CCK-8 was used to measure cell proliferation and cell cycles were analyzed using flow cytometry. A dual luciferase reporter gene assay was used to confirm the downstream targets of miR-0308-3p. RESULTS: In total, there were 34 overlapping miRNAs in both our miRNA-seq data and the TCGA database. We found two overlapping miRNAs in both the HBV-positive HCC samples and the TCGA database, and 205 novel pre-miRNA sequences were predicted. miR-522 and miR-523 were markedly overexpressed in HBV-positive HCC and were associated with a significantly poorer long-term prognosis (miR-522, HR 2.19, 95% CI 1.33-3.6, p = 0.0015; miR-523HR 1.5, 95% CI 1-2.44, p = 0.0047). Of note, we found that the novel miR-0308-3p was markedly downregulated in HBV-positive HCC samples and HCC cancer cell lines compared with HBV-negative HCC samples and adjacent normal hepatic tissue. Moreover, elevated expression of miR-0308-3p was found to inhibit proliferation of cancer cells by promoting G1/S cell cycle arrest but did not influence the apoptosis of cancer cells. A dual luciferase reporter activity assay identified that miR-0308-3p acted directly on the target sequence of the CDK6 and Cyclin D1 mRNA 3'UTR to suppress CDK6 and Cyclin D1 expression. CONCLUSIONS: MiR-0308-3p upregulation dramatically suppressed HCC cell proliferation and induced G1/S cell cycle arrest by directly targeting CDK6/Cyclin D1. These findings reveal a novel molecular mechanism for activation of G1/S arrest in HCC and may prove clinically useful for developing new therapeutic targets.

Analysis of mRNA Expression Patterns in Peripheral Blood Cells of 3 Patients With Cancer After the First Fraction of 2 Gy Irradiation: An Integrated Case Report and Systematic Review.

BACKGROUND: Radiation therapy induces acute and chronic radiological toxicity, in particular hematological toxicity (HT). This study aimed to explore the mechanistic clue and potential predictors at the messenger RNA (mRNA) level. MATERIALS AND METHODS: Peripheral blood was collected from 3 patients with cervical cancer (CC), nasopharynx cancer (NC), and tongue cancer (TC) after the first 2 Gy fraction of radiotherapy (RT). High-throughput sequencing was used to assess mRNA profiles. RESULTS: Eleven genes, such as ALAS2(5-aminolevulinate synthase), SLC4A1(solute carrier family 4 member 1), HBG2(hemoglobin subunit gamma 2), TNFAIP3 (TNF α-induced protein 3), PER1 (period circadian clock 1), CCDC136 (coiled-coil domain containing 136), C9orf84 (chromosome 9 open reading frame 84), IL1B (interleukin 1ß), FOSB (FosB protooncogene), NR4A2 (nuclear receptor subfamily 4), PARP15 (polymerase family member 15), had overlapping expression changes in all 3 cancers of which 3 (ALAS2, FOSB, and HBG2) are suggested as potential predictors for the early diagnosis of HT after RT. CONCLUSIONS: ALAS2, FOSB, and HBG2 may be useful predictors of HT in patients after RT. Eleven overlapping expression mRNAs among 3 cancers might be potential predictors for early diagnosis of radiation toxicity in patients.

siRNA-mediated silencing of Cockayne Cyndrome group B gene potentiates radiation-induced apoptosis and antiproliferative effect in HeLa cells.

BACKGROUND: Cockayne syndrome (CS) is a rare human genetic disorder characterized by increased UV sensitivity, developmental abnormalities and premature aging. Cells isolated from individuals with CS have a defect in transcription-coupled DNA repair. Despite the repair defect, there is no any increased risk of spontaneous or UV-induced cancer for CS individuals. The strategy of RNA interfering was used here to explore the potential radiosensitizing and anticancer activity of targeting CS group B (CSB) gene. METHODS: The vectors encoding CSB-specific siRNAs were constructed by inserting duplex siRNA encoding oligonucleotides into the plasmid P(silencer TM 3.1). The cell lines expressing the CSB-siRNA were generated from HeLa cells transfected with the above vectors. Colony-forming ability was used to assay cell survival. Cell cycle was analyzed by FACScan flow cytometry. The apoptosis was measured by detecting the accumulation of sub-G(1) population as well as by fluorescence staining assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to semi-quantify mRNA expression. Protein level was detected by Western blotting analysis. RESULTS: Two constructs encoding CSB-specific siRNA were generated, both of them resulted in remarkable suppression on CSB expression in HeLa cells, and led to an increased sensitivity to (gamma-ray and UV light. siRNA-mediated silencing of CSB decreased cell proliferation rate, increased spontaneous apoptosis as well as the occurrence of UV- or cisplatin-induced apoptosis by 2 to 3.5 fold. A significant S phase blockage and a remarkable reduction of G(1) population were induced in control HeLa cells at 18 hours after being exposed to 10 J/m(2) of UV light. The S phase blockage was also observed in UV-irradiated CSB-siRNA transfected HeLa cells, but the extent of increased S phase population was lower than that in the UV-irradiated control cells. No or a relative weak reduction on G(1) phase population was observed in UV-irradiated CSB-siRNA transfected HeLa cells. In addition, siRNA-mediated silencing of CSB promoted the elimination of G(2)/M phase cells after UV light radiation. CONCLUSIONS: siRNA-mediated silencing of CSB causes cells to proliferate more slowly, sensitize cells to genotoxicants, and modify UV radiation-induced cell cycle changes. siRNA-mediated inactivation of CSB could be an attractive strategy for ameliorating cancer therapy, which can be fulfilled via the combination of gene therapy and sensitization of radiotherapy or chemotherapy.