High levels of cadmium altered soil archaeal activity, assembly, and co-occurrence network in volcanic areas.

Soil microbial communities are essential to biogeochemical cycles. However, the responses of microorganisms in volcanic soil with high heavy metal levels remain poorly understood. Here, two areas with high levels of cadmium (Cd) from the same volcano were investigated to determine their archaeal composition and assembly. In this study, the Cd concentrations (0.32-0.38 mg/ kg) in the volcanic soils exceeded the standard risk screening values (GB15618-2018) and correlated with archaeal communities strongly (P < 0.05). Moreover, the area with elevated levels of Cd (periphery) exhibited a greater diversity of archaeal species, albeit with reduced archaeal activity, compared to the area with lower levels of Cd (center). Besides, stochastic processes mainly governed the archaeal communities. Furthermore, the co-occurrence network was simplest in the periphery. The proportion of positive links between taxa increased positively with Cd concentration. Moreover, four keystone taxa (all from the family Nitrososphaeraceae) were identified from the archaeal networks. In its entirety, this study has expanded our comprehension of the variations of soil archaeal communities in volcanic areas with elevated cadmium levels and serves as a point of reference for the agricultural development of volcanic soils in China.

Key Targets and Molecular Mechanisms of the Fat-soluble Components of Ginseng for Lung Cancer Treatment.

OBJECTIVE: To analyze the regulatory effects and key targets of the fat-soluble components of ginseng in lung cancer. METHODS: Gas chromatography-mass spectrometry and the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform were used to analyze and identify the fat-soluble components of ginseng. Network pharmacology was used to analyze the therapeutic targets of the fat-soluble components of ginseng in lung cancer and screen key proteins. In vitro assays were conducted to verify the effects of the active fat-soluble components of ginseng on proliferation and apoptosis in lung cancer cells and to verify the regulation of key proteins. RESULTS: Ten active fat-soluble components of ginseng were screened for follow-up. Network pharmacology showed 33 overlapping targets between the active fat-soluble components of ginseng and lung cancer, and functional enrichment of the targets showed involvement of response to nitrogen, hormone response, membrane raft, and positive regulation of external stimulus. Pathway enrichment analysis showed vascular endothelial growth factor (VEGF) signaling, adipocyte lipolysis regulation, chronic myelogenous leukemia, endocrine resistance, and NSCLC-related pathways. A protein-protein interaction network was constructed, and the top 10 targets were selected in accordance with their scores. Ultimately, five target genes (EGFR, KDR, MAPK3, PTPN11, and CTNNB1) were selected in combination with literature mining for subsequent experimental verification. Proliferation assays showed that the growth of lung cancer cells was significantly decreased in a concentration-dependent manner in the fat-soluble components of ginseng intervention group compared with controls. Flow cytometry showed that active fat-soluble components of ginseng promoted apoptosis in a concentration-dependent manner in lung cancer cells. Western blot and quantitative real-time PCR showed that levels of the five key proteins and mRNAs were significantly decreased in the intervention group; furthermore, histone protein and mRNA levels were significantly higher in the high-concentration intervention group compared with the low-concentration group. CONCLUSION: The active fat-soluble components of ginseng inhibited the growth of lung cancer cells and promoted apoptosis. The underlying regulatory mechanisms may be related to signaling pathways involving EGFR, KDR, MAPK3, PTPN11, and CTNNB1.

Key Targets and Molecular Mechanisms of Active Volatile Components of Rabdosia rubescens in Gastric Cancer Cells.

OBJECTIVE: To examine the effect and mechanism of volatile components of Rabdosia rubescens on gastric cancer. METHODS: Gas chromatography-mass spectrometry was used to detect and identify the volatile components of R. rubescens. The network pharmacology method was used to analyze the targets of volatile components of R. rubescens in gastric cancer and to reveal their molecular mechanisms. The effects of volatile components of R. rubescens on gastric cancer cells were verified by biological experiments. RESULTS: Thirteen volatile components of R. rubescens were selected as pharmacologically active components. The 13 active components had 83 targets in gastric cancer, and a Traditional Chinese Medicine-component-targets gastric cancer network was successfully constructed. Five core targets were obtained: TNF, IL1B, MMP9, PTGS2 and CECL8. The volatile components inhibited the proliferation of gastric cancer cells in a concentration-dependent manner and promoted the apoptosis of gastric cancer cells. The volatile components reduced the levels of TNF, IL1B, MPP9, and PTGS2 in a concentration-dependent manner. CONCLUSION: Our study demonstrates the effects of volatile components in R. rubescens on gastric cancer and provides preliminary findings on their mechanisms of action.

Construction and validation of a novel prognostic model for thyroid cancer based on N7-methylguanosine modification-related lncRNAs.

BACKGROUND: To construct and verify a novel prognostic model for thyroid cancer (THCA) based on N7-methylguanosine modification-related lncRNAs (m7G-lncRNAs) and their association with immune cell infiltration. METHODS: In this study, we identified m7G-lncRNAs using co-expression analysis and performed differential expression analysis of m7G-lncRNAs between groups. We then constructed a THCA prognostic model, performed survival analysis and risk assessment for the THCA prognostic model, and performed independent prognostic analysis and receiver operating characteristic curve analyses to evaluate and validate the prognostic value of the model. Furthermore, analysis of the regulatory relationship between prognostic differentially expressed m7G-related lncRNAs (PDEm7G-lncRNAs) and mRNAs and correlation analysis of immune cells and risk scores in THCA patients were carried out. RESULTS: We identified 29 N7-methylguanosine modification-related mRNAs and 116 differentially expressed m7G-related lncRNAs, including 87 downregulated and 29 upregulated lncRNAs. Next, we obtained 8 PDEm7G-lncRNAs. A final optimized model was constructed consisting of 5 PDEm7G-lncRNAs (DOCK9-DT, DPP4-DT, TMEM105, SMG7-AS1 and HMGA2-AS1). Six PDEm7G-lncRNAs (DOCK9-DT, DPP4-DT, HMGA2-AS1, LINC01976, MID1IP1-AS1, and SMG7-AS1) had positive regulatory relationships with 10 PDEm7G-mRNAs, while 2 PDEm7G-lncRNAs (LINC02026 and TMEM105) had negative regulatory relationships with 2 PDEm7G-mRNAs. Survival curves and risk assessment predicted the prognostic risk in both groups of patients with THCA. Forest maps and receiver operating characteristic curves were used to evaluate and validate the prognostic value of the model. Finally, we demonstrated a correlation between different immune cells and risk scores. CONCLUSION: Our results will help identify high-risk or low-risk patients with THCA and facilitate early prediction and clinical intervention in patients with high risk and poor prognosis.

Genome-Wide Identification of Direct Targets of ZjVND7 Reveals the Putative Roles of Whole-Genome Duplication in Sour Jujube in Regulating Xylem Vessel Differentiation and Drought Tolerance.

The effects of whole-genome duplication span multiple levels. Previous study reported that the autotetraploid sour jujube exhibited superior drought tolerance than diploid. However, the difference in water transport system between diploids and autotetraploids and its mechanism remain unclear. Here, we found the number of xylem vessels and parenchyma cells in autotetraploid sour jujube increased to nearly twice that of diploid sour jujube, which may be closely related to the differences in xylem vessel differentiation-related ZjVND7 targets between the two ploidy types. Although the five enriched binding motifs are different, the most reliable motif in both diploid and autotetraploid sour jujube was CTTNAAG. Additionally, ZjVND7 targeted 236 and 321 genes in diploids and autotetraploids, respectively. More identified targeted genes of ZjVND7 were annotated to xylem development, secondary wall synthesis, cell death, cell division, and DNA endoreplication in autotetraploids than in diploids. SMR1 plays distinct roles in both proliferating and differentiated cells. Under drought stress, the binding signal of ZjVND7 to ZjSMR1 was stronger in autotetraploids than in diploids, and the fold-changes in the expression of ZjVND7 and ZjSMR1 were larger in the autotetraploids than in the diploids. These results suggested that the targeted regulation of ZjVND7 on ZjSMR1 may play valuable roles in autotetraploids in the response to drought stress. We hypothesized that the binding of ZjVND7 to ZjSMR1 might play a role in cell division and transdifferentiation from parenchyma cells to vessels in the xylem. This regulation could prolong the cell cycle and regulate endoreplication in response to drought stress and abscisic acid, which may be stronger in polyploids.

Identification of Benign and Malignant Lung Nodules in CT Images Based on Ensemble Learning Method.

BACKGROUND AND OBJECTIVE: Under the background of urgent need for computer-aided technology to provide physicians with objective decision support, aiming at reducing the false positive rate of nodule CT detection in pulmonary nodules detection and improving the accuracy of lung nodule recognition, this paper puts forward a method based on ensemble learning to distinguish between malignant and benign pulmonary nodules. METHODS: Firstly, trained on a public data set, a multi-layer feature fusion YOLOv3 network is used to detect lung nodules. Secondly, a CNN was trained to differentiate benign from malignant pulmonary nodules. Then, based on the idea of ensemble learning, the confidence probability of the above two models and the label of the training set are taken as data features to build a Logistic regression model. Finally, two test sets (public data set and private data set) were tested, and the confidence probability output by the two models was fused into the established logistic regression model to determine benign and malignant pulmonary nodules. RESULTS: The YOLOv3 network was trained to detect chest CT images of the test set. The number of pulmonary nodules detected in the public and private test sets was 356 and 314, respectively. The accuracy, sensitivity and specificity of the two test sets were 80.97%, 81.63%, 78.75% and 79.69%, 86.59%, 72.16%, respectively. With CNN training pulmonary nodules benign and malignant discriminant model analysis of two kinds of test set, the result of accuracy, sensitivity and specificity were 90.12%, 90.66%, 89.47% and 88.57%, 85.62%, 90.87%, respectively. Fused model based on YOLOv3 network and CNN is tested on two test sets, and the result of accuracy, sensitivity and specificity were 93.82%, 94.85%, 92.59% and 92.31%, 92.68%, 91.89%, respectively. CONCLUSION: The ensemble learning model is more effective than YOLOv3 network and CNN in removing false positives, and the accuracy of the ensemble. Learning model is higher than the other two networks in identifying pulmonary nodules.

Establishment of a Lung Cancer Discriminative Model Based on an Optimized Support Vector Machine Algorithm and Study of Key Targets of Wogonin in Lung Cancer.

An optimized support vector machine model was used to construct a lung cancer diagnosis model based on serological indicators, and a molecular regulation model of Wogonin, a component of Scutellaria baicalensis, was established. Serological indexes of patients were collected, the grid search method was used to identify the optimal penalty coefficient C and parameter g of the support vector machine model, and the benign and malignant auxiliary diagnosis model of isolated pulmonary nodules based on serological indicators was established. The regulatory network and key targets of Wogonin in lung cancer were analyzed by network pharmacology, and key targets were detected by western blot. The relationship between serological susceptibility genes and key targets of Wogonin was established, and the signaling pathway of Wogonin regulating lung cancer was constructed. After support vector machine parameter optimization (C = 90.597, g = 32), the accuracy of the model was 90.8333%, with nine false positives and two false negative cases. Ontology functional analysis of 67 common genes between Wogonin targets and lung cancer-related genes showed that the targets were associated with biological processes involved in peptidye-serine modification and regulation of protein kinase B signaling; cell components in the membrane raft and chromosomal region; and molecular function in protein serine/threonine kinase activity and heme binding. Kyoto Encyclopedia of Genes and Genomes analysis showed that the regulation pathways involved the PI3K-Akt signaling pathway, ERBB signaling pathway, and EGFR tyrosine kinase inhibitor resistance. In vitro analyses using lung cancer cells showed that Wogonin led to significantly increased levels of cleaved caspase-3 and Bad and significantly decreased Bcl-2 expression in a concentration-dependent manner. ErbB4 expression also significantly decreased in lung cancer cells after treatment with Wogonin. A regulatory network of Wogonin regulating lung cancer cell apoptosis was constructed, including the participation of serological susceptibility genes. There is a certain regulatory effect between the serological indexes that can be used in the diagnosis of lung cancer and the key targets of Chinese herbal medicine treatment of lung cancer, which provides a new idea for the diagnosis, treatment and prognosis of clinical lung cancer.

Multiple responses contribute to the enhanced drought tolerance of the autotetraploid Ziziphus jujuba Mill. var. spinosa.

BACKGROUND: Polyploid plants often exhibit enhanced stress tolerance. The underlying physiological and molecular bases of such mechanisms remain elusive. Here, we characterized the drought tolerance of autotetraploid sour jujube at phenotypic, physiological and molecular levels. RESULTS: The study findings showed that the autotetraploid sour jujube exhibited a superior drought tolerance and enhanced regrowth potential after dehydration in comparison with the diploid counterpart. Under drought stress, more differentially expressed genes (DEGs) were detected in autotetraploid sour jujube and the physiological responses gradually triggered important functions. Through GO enrichment analysis, many DEGs between the diploid and autotetraploid sour jujube after drought-stress exposure were annotated to the oxidation-reduction process, photosystem, DNA binding transcription factor activity and oxidoreductase activity. Six reactive oxygen species scavenging-related genes were specifically differentially expressed and the larger positive fold-changes of the DEGs involved in glutathione metabolism were detected in autotetraploid. Consistently, the lower O2- level and malonaldehyde (MDA) content and higher antioxidant enzymes activity were detected in the autotetraploid under drought-stress conditions. In addition, DEGs in the autotetraploid after stress exposure were significantly enriched in anthocyanin biosynthesis, DNA replication, photosynthesis and plant hormone, including auxin, abscisic acid and gibberellin signal-transduction pathways. Under osmotic stress conditions, genes associated with the synthesis and transport of osmotic regulators including anthocyanin biosynthesis genes were differentially expressed, and the soluble sugar, soluble protein and proline contents were significantly higher in the autotetraploid. The higher chlorophyll content and DEGs enriched in photosynthesis suggest that the photosynthetic system in the autotetraploid was enhanced compared with diploid during drought stress. Moreover, several genes encoding transcription factors (TFs) including GRAS, Bhlh, MYB, WRKY and NAC were induced specifically or to higher levels in the autotetraploid under drought-stress conditions, and hub genes, LOC107403632, LOC107422279, LOC107434947, LOC107412673 and LOC107432609, related to 18 up-regulated transcription factors in the autotetraploid compared with the diploid were identified. CONCLUSION: Taken together, multiple responses contribute to the enhanced drought tolerance of autotetraploid sour jujube. This study could provide an important basis for elucidating the mechanism of tolerance variation after the polyploidization of trees.

Network Pharmacology for Analyzing the Key Targets and Potential Mechanism of Wogonin in Gliomas.

Objective: To analyze the key targets and potential mechanisms underlying the volatile components of Scutellaria baicalensis Georgi acting on gliomas through network pharmacology combined with biological experiments. Methods: We have extracted the volatile components of Scutellaria baicalensis by gas chromatography-mass spectrometry (GC-MS) and determined the active components related to the onset and development of gliomas by combining the results with the data from the Traditional Chinese Medicine Systems Pharmacology database. We screened the same targets for the extracted active components and gliomas through network pharmacology and then constructed a protein-protein interaction network. Using a Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, we analyzed the protein effects and regulatory pathways of the common targets. Lastly, we employed ELISA and Western blot in verifying the key targets in the regulatory pathway. Results: We ultimately determined that the active component in S. baicalensis Georgi related to the onset and development of gliomas was Wogonin. The results of the network pharmacology revealed 85 targets for glioma and Wogonin. We used gene ontology to analyze these target genes and found that they involved 30 functions, such as phosphatidylinositol phosphokinase activation, while the KEGG analysis showed that there were 10 regulatory pathways involved. Through the following analysis, we found that most of the key target genes are distributed in the PI3K-Akt and interleukin 17 signaling pathways. We then cultured U251 glioma cells for the experiments. Compared with the control group, no significant change was noted in the caspase-3 expression; however, cleaved caspase-3 expression increased significantly and was dose-dependent on Wogonin. The expression of Bad and Bcl-2 with 25 µM of Wogonin has remained unchanged, but when the Wogonin dose was increased to 100 µM, the expression of Bad and Bcl-2 was noted to change significantly (Bad was significantly upregulated, while Bcl-2 was significantly downregulated) and was dose-dependent on Wogonin. The ELISA results showed that, compared with the control group, the secretion of tumor necrosis factor alpha, IL-1ß, and IL-6 decreased as the Wogonin concentration increased. Tumor necrosis factor alpha downregulation had no significant dose-dependent effect on Wogonin, the inhibitory effect of 25 µM of Wogonin on IL-6 was not significant, and IL-1ß downregulation had a significant dose-dependent effect on Wogonin. Conclusion: Wogonin might promote the apoptosis of glioma cells by upregulating proapoptotic factors, downregulating antiapoptotic factors, and inhibiting the inflammatory response, thereby inhibiting glioma progression.

Comparative study on different therapies on patients with primary central nervous system lymphoma.

OBJECTIVE: To compare and analyze three therapies on patients with primary central nervous system lymphoma (PCNSL), aiming to provide evidences for future treatment and prognosis. METHODS: Clinical data of 26 cases of PCNSL with normal immune system confirmed by postoperative pathology were retrospectively analyzed. Among them there were six cases with operation only, nine cases with operation and radiotherapy, and 11 cases with operation, radiotherapy and chemotherapy, and their survival rate was compared as well. RESULTS: The survival time of patients with operation only, operation combined with radiotherapy and operation combined with radiotherapy and chemotherapy was 6-11 months, 15-24 months and 24-51 months, respectively. And their median survival time was only nine months, 21 months and 38 months, respectively. CONCLUSIONS: Operation combined with radiotherapy and chemotherapy can dramatically extend PCNSL patients' survival time, therefore, it can be regarded as the first-line therapy.

Construction of a mitochondrial-associated protein DRP1 and a lung cancer-associated protein Erbb4 combined regulatory network.

OBJECTIVE: This research was to establish a mitochondrial-related Drp1 gene and a lung cancer-related Erbb4 gene to participate in the regulatory network of lung cancer cell apoptosis, and to provide theoretical support for mitochondria to participate in tumor regulation. METHOD: The GO and KEGG methods were used to construct the regulatory networks of lung cancer related Drp1 and Erbb4 proteins that involved in the apoptosis of tumor cells, and to combine with the Bayesian network theory to screen out the largest possible action path acting on this network; The information about Drp1 in Oncomine database was collected, and the data in current database were analyzed twice. The role of Drp1 in lung cancer was meta-analyzed. RESULT: A regulatory network of Drp1 and Erbb4 involved in the apoptosis of tumor cells was successfully constructed; the optimal pathway was optimized using Bayesian theory; a total of 446 different types of research results were collected in the Oncomine database, of which there were 18 studies with statistical differences in Drp1 expression, 13 studies with increased Drp1's expression, and 5 studies with decreased expression. Compared with the control group, Drp1 was expressed in lung cancer tissues highly (P < 0.05). CONCLUSION: Establishment and optimization of mitochondrial-related Drp1 and tumor-related Erbb4 genes involved in the regulation of apoptosis of cancer cells. It was proposed that Drp1 was expressed in lung cancer tissues highly through in-depth excavation of tumor-associated gene information in the Oncomine gene chip database.

An Assessment of Dietary Exposure to Cadmium in Residents of Guangzhou, China.

Cadmium and its compounds are human carcinogens with severe organ toxicity, and their contamination of agricultural soil in China has been frequently reported; however, the dietary exposure to cadmium in residents and the relevant health risk have seldom been reported. In this study, the concentration of cadmium in various types of food collected from 2013 to 2015 were analyzed using graphite furnace atomic absorption spectrometry, and the dietary exposure to cadmium assessed based on a dietary survey in 2976 Guangzhou residents. In total, 3074 out of 4039 food samples had cadmium levels above the limit of detection. The mean ± standard deviation (50th, 95th percentile) cadmium content in all samples was 159.0 ± 112.7 (8.6, 392.4) µg/kg, with levels ranging from 1.0 to 7830 µg/kg. Using the mean cadmium concentrations, the average monthly dietary exposure of Guangzhou residents to cadmium was 14.4 (µg/kg body weight (BW), accounting for 57.6% of the provisional tolerable monthly intake (PTMI). Rice, laver, vegetables, and live aquatic products were the main sources of cadmium intake, on average accounting for 89% of the total value. The dietary cadmium exposure in high consumers (95th percentile food consumption) was 41.0 µg/kg·BW/month, accounting for 163% of the PTMI. Additionally, dietary cadmium exposure at mean consumption but high cadmium food concentration (95th percentile) was 32.3 µg/kg·BW/month, corresponding to 129% of the PTMI. The level of dietary exposure to cadmium in most Guangzhou residents was within the safety limit, thus increased health risk from dietary cadmium exposure is low at present. However, continued efforts by local governments to monitor the levels of cadmium in the four main food categories contributing to exposure are necessary.

Sumoylation pathway is required to maintain the basal breast cancer subtype.

The TFAP2C/AP-2γ transcription factor regulates luminal breast cancer genes, and loss of TFAP2C induces epithelial-mesenchymal transition. By contrast, the highly homologous family member, TFAP2A, lacks transcriptional activity at luminal gene promoters. A detailed structure-function analysis identified that sumoylation of TFAP2A blocks its ability to induce the expression of luminal genes. Disruption of the sumoylation pathway by knockdown of sumoylation enzymes, mutation of the SUMO-target lysine of TFAP2A, or treatment with sumoylation inhibitors induced a basal-to-luminal transition, which was dependent on TFAP2A. Sumoylation inhibitors cleared the CD44(+/hi)/CD24(-/low) cell population characterizing basal cancers and inhibited tumor outgrowth of basal cancer xenografts. These findings establish a critical role for sumoylation in regulating the transcriptional mechanisms that maintain the basal cancer phenotype.

Stacking resistance to crown gall and nematodes in walnut rootstocks.

BACKGROUND: Crown gall (CG) (Agrobacterium tumefaciens) and the root lesion nematodes (RLNs) (Pratylenchus vulnus) are major challenges faced by the California walnut industry, reducing productivity and increasing the cost of establishing and maintaining orchards. Current nematode control strategies include nematicides, crop rotation, and tolerant cultivars, but these methods have limits. Developing genetic resistance through novel approaches like RNA interference (RNAi) can address these problems. RNAi-mediated silencing of CG disease in walnut (Juglans regia L.) has been achieved previously. We sought to place both CG and nematode resistance into a single walnut rootstock genotype using co-transformation to stack the resistance genes. A. tumefaciens, carrying self-complimentary iaaM and ipt transgenes, and Agrobacterium rhizogenes, carrying a self-complimentary Pv010 gene from P. vulnus, were used as co-transformation vectors. RolABC genes were introduced by the resident T-DNA in the A. rhizogenes Ri-plasmid used as a vector for plant transformation. Pv010 and Pv194 (transgenic control) genes were also transferred separately using A. tumefaciens. To test for resistance, transformed walnut roots were challenged with P. vulnus and microshoots were challenged with a virulent strain of A. tumefaciens. RESULTS: Combining the two bacterial strains at a 1:1 rather than 1:3 ratio increased the co-transformation efficiency. Although complete immunity to nematode infection was not observed, transgenic lines yielded up to 79% fewer nematodes per root following in vitro co-culture than untransformed controls. Transgenic line 33-3-1 exhibited complete crown gall control and 32% fewer nematodes. The transgenic plants had thicker, longer roots than untransformed controls possibly due to insertion of rolABC genes. When the Pv010 gene was present in roots with or without rolABC genes there was partial or complete control of RLNs. Transformation using only one vector showed 100% control in some lines. CONCLUSIONS: CG and nematode resistance gene stacking controlled CG and RLNs simultaneously in walnuts. Silencing genes encoding iaaM, ipt, and Pv010 decrease CG formation and RLNs populations in walnut. Beneficial plant genotype and phenotype changes are caused by co-transformation using A. tumefaciens and A. rhizogenes strains. Viable resistance against root lesion nematodes in walnut plants may be accomplished in the future using this gene stacking technology.

RNA interference targeting Akt promotes apoptosis in hypoxia-exposed human neuroblastoma cells.

Overactivation of the PI3 kinase/Akt pathway plays an essential role in the development and progression of various tumors. Akt is a key component of this pathway and hyperactivated in different tumors including neuroblastoma and glioma. In the present study, we tested the therapeutic efficacy of siRNA targeting Akt in inducing apoptotic cell death in NBFL cells (a human neuroblastoma cell line) subjected to anoxia/reoxygenation (A/R), a process that has been shown to modulate growth and progression of malignant tumors. We observed that siRNA targeting Akt effectively induced apoptotic cell death in NBFL cells (as determined by TUNEL assay and activated caspase-3 immunoreactivity) under normoxic conditions, an effect that was greatly enhanced under conditions of A/R. These findings underscore the importance of Akt signaling in promoting survival of neuroblastoma cells and may have potential therapeutic applications.

HIF-1alpha has an anti-apoptotic effect in human airway epithelium that is mediated via Mcl-1 gene expression.

Hypoxia-inducible factor-1alpha (HIF-1alpha) and myeloid cell leukemia-1 (Mcl-1) proteins have been shown to regulate apoptosis in some cell systems but have not been studied in this context in airway epithelium. Using a model of anoxia/reoxygenation (A/R), the present study employed RNA interference (RNAi) targeting HIF-1alpha and Mcl-1 to evaluate their possible anti-apoptotic effects on HBE1 cells, an immortalized human bronchial epithelial cell line. The cells were either cultured under normoxic conditions or were transfected with small interfering RNA (siRNA) duplexes targeting HIF-1alpha or Mcl-1 mRNA and then immediately exposed to A/R. As controls, non-transfected HBE1 cells and cells transfected with scrambled RNA duplexes were subjected to A/R. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and RNAi was assessed by knockdown of HIF-1alpha and Mcl-1 mRNA and protein expression using real-time quantitative RT-PCR (Q-PCR), immunohistochemistry, and Western blots. HBE1 cells transfected with siRNA duplexes targeting either HIF-1alpha or Mcl-1 and subjected to A/R manifested considerable apoptosis, a finding not observed in either non-transfected cells or cells transfected with scrambled RNA duplexes. Specific knockdown of mRNA and protein expression by RNAi in HBE1 cells after A/R was shown for siRNA duplexes targeting either HIF-1alpha or Mcl-1. Unexpectedly, knockdown of HIF-1alpha induced parallel knockdown of Mcl-1 mRNA and protein expression, whereas Mcl-1 knockdown had no noticeable effect on HIF-1alpha expression. Thus, although both of these proteins were shown to be anti-apoptotic, the action of HIF-1alpha appeared to be mediated in part via Mcl-1.

Hemorrhagic shock induces differential gene expression and apoptosis in mouse liver.

Comprehensive knowledge of the gene expression changes induced by hemorrhage in vital organs will greatly improve prognosis and therapy. Therefore, we used a mouse model of non-resuscitated hemorrhagic shock to study the pattern of stress-induced genes in liver at 1, 4, and 24 h following surgery. Hepatic injury was confirmed by assessment of liver injury markers and apoptotic cell death. We found that a variety of stress-regulated genes were differentially expressed, including seven genes that have not been reported previously as being regulated by hemorrhagic shock: ATF-2, alphaB-crystallin, GADD45, GADD45beta, Mdm2, p21Waf1, and TRPM-2. The changes in mRNA levels of the transcription factors AP-1, Egr-1, HSF-1, and NF-kappaB were transient but protein expression was noticeable at later time points. Our findings show that oxidative stress causes immediate upregulation of genes involved in a variety of cellular defense pathways. Complex interactions among them might determine the ultimate fate of the cell.

Akt phosphorylation and kinase activity are down-regulated during hibernation in the 13-lined ground squirrel.

Hibernation in mammals is a reversible state of suspended animation associated with tolerance to an otherwise lethal reduction of core body temperature and metabolism. An integral aspect of hibernation is tolerance to a profound decrease of cerebral perfusion. Identification of regulatory mechanisms that control hibernation in ground squirrels can guide efforts to develop improved treatment for stroke and brain trauma. In this study, we show in multiple tissues that S473 phosphorylation of Akt (Protein kinase B), a phosphatidylinositol-3 kinase-regulated serine/threonine kinase, was significantly reduced (P<0.001) as was its kinase activity (P=0.023) in the 13-lined ground squirrel, Spermophilus tridecemlineatus, during hibernation. T308 phosphorylation of Akt was relatively preserved. Brain immunohistochemical staining confirmed these results. In hibernating animals, reduction of immunoreactive phospho (S473)-Akt was noted throughout the brain. Akt is a key molecule in the insulin/insulin-like growth factor signal transduction pathway, which plays a critical role in the balance between survival and apoptosis. The data presented here raise the possibility that down-regulation of Akt phosphorylation plays a regulatory role in hibernation. This would resemble dauer larva formation in Caenorhabditis elegans where Akt inhibition is associated with energy conservation, fat storage, expression of antioxidant enzymes and growth arrest.

Asbestos inhalation induces tyrosine nitration associated with extracellular signal-regulated kinase 1/2 activation in the rat lung.

Nitration of proteins by peroxynitrite (ONOO-) has been shown to critically alter protein function in vitro. We have shown previously that asbestos inhalation induced nitrotyrosine formation, a marker of ONOO- production, in the rat lung. To determine whether asbestos-induced protein nitration may affect mitogen-activated protein kinase (MAPK) signaling pathways, lung lysates from crocidolite and chrysotile asbestos-exposed rats and from sham-exposed rats were immunoprecipitated with anti-nitrotyrosine antibody, and captured proteins were subjected to Western blotting with anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibodies. Both types of asbestos inhalation induced significantly greater phosphorylation of ERK1/2 in rat lung lysates than was noted after sham exposure. Phosphorylated ERK proteins co-immunoprecipitated with nitrotyrosine. Moreover, in MAPK functional assays using Elk-1 substrate, immunoprecipitated phospho-ERK1/2 in lung lysates from both crocidolite-exposed and chrysotile-exposed rats demonstrated significantly greater phosphorylation of Elk-1 than was noted after sham exposure. Asbestos inhalation also induced ERK phosphorylation in bronchoalveolar lavage cells. Lung sections from rats exposed to crocidolite or chrysotile (but not from sham-exposed rats nor from rats exposed to "inert" carbonyl iron particles) demonstrated strong immunoreactivity for nitrotyrosine and phospho-ERK1/2 in alveolar macrophages and bronchiolar epithelium. These findings suggest that asbestos fibers may activate the ERK signaling pathway by generating ONOO- or other nitrating species that induce tyrosine nitration and phosphorylation of critical signaling molecules.