Although anomalies in the topological architecture of whole-brain connectivity have been found to be associated with Alzheimer’s disease (AD), our understanding about the progression of AD in a functional connectivity (FC) perspective is still rudimentary and few study has explored the function-structure relations in brain networks of AD patients. By using resting-state functional MRI (fMRI), this study firstly investigated organizational alternations in FC networks in 12 AD patients, 15 amnestic mild cognitive impairment (aMCI) patients, and 14 age-matched healthy aging subjects and found that all three groups exhibit economical small-world network properties. Nonetheless, we found a decline of the optimal architecture in the progression of AD, represented by a more localized modular organization with less efficient local information transfer. Our results also show that aMCI forms a boundary between normal aging and AD and represents a functional continuum between healthy aging and the earliest signs of dementia. Moreover, we revealed a dissociated relationship between the overall FC and structural connectivity (SC) in AD patients. In this study, diffusion tensor imaging tractography was used to map the structural network of the same individuals. The decreased FC-SC coupling may be indicative of more stringent and less dynamic brain function in AD patients. Our findings provided insightful implications for understanding the pathophysiological mechanisms of brain dysfunctions in aMCI and AD patients and demonstrated that functional disorders can be characterized by multimodal neuroimaging-based metrics.
To investigate a possible methodology of exploiting herbal medicine and design polytherapy for the treatment of non-alcoholic fatty liver disease (NAFLD), we have made use of Cichorium glandulosum
Boiss et Huet (CG), a traditional Chinese herbal medicine that has been proven to be effective in treating hepatic diseases. Here, we report that the extract of CG effectively reduced lipid accumulation under conditions of lipid overloading in vivo and in vitro (in a rat high-fat diet model and a hepG2 cell model of free fatty acid treatment). CG extract also protected hepatocytes from injury and inflammation to aid its lipid-lowering properties (in a rat high-fat diet model and a L02 cell model of acetaminophen treatment). Serum chemistry analysis accompanied by in vitro drug screening confirmed that CG-4, CG-10 and CG-14 are the lipo-effective components of CG. Western blotting analysis revealed that these components can regulate key lipid targets at the molecular level, including CD36, FATP5 and PPAR-α, thus the lipid oxidation and lipid absorption pathways. Finally, we adopted the experimental design and statistical method to calculate the best combination proportion (CG-4: CG-10: CG-14 = 2.065: 1.782: 2.153) to optimize its therapeutic effect.
Somatic LKB1 mutations are found in lung adenocarcinomas at different frequencies in Caucasian and East Asian (Japanese and Korean) populations. This study was designed to characterize the frequency of LKB1 mutations, their relationship to EGFR and KRAS mutations, and their associated clinicopathologic characteristics in Chinese patients.
Two hundred thirty-nine lung adenocarcinomas consecutively collected from October 2007 to July 2009 were dissected into 3 to 4 small (3 mm) pieces for histopathological analyses of tumor content. Genomic DNA and/or cDNA from 86 samples with more than 70% tumor content were used for sequencing of LKB1 (exons 1–9), EGFR (exons 18–21), and KRAS (exon 2). LKB1 germline mutation status was determined by sequencing of genomic DNA from matched histologically distant lung tissues that are histologically normal.
6.9% of lung adenocarcinomas harbored LKB1 somatic mutations. A total of 10.5% of patients had an LKB1 germline polymorphism, F354L. Interestingly, in two of these patients, tumors displayed loss of heterozygosity at this allele. EGFR kinase domain and KRAS mutations were found in 66.3% and 2.3% of Chinese lung adenocarcinomas, respectively. Concurrent LKB1 and EGFR somatic mutations were observed in one patient. Both KRAS-mutant tumors harbored LKB1 mutations.
These data provide important clinical and molecular characteristics of lung adenocarcinomas from Chinese patients.
Chinese lung adenocarcinoma; LKB1; EGFR; KRAS; Mutation
Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.
Alternative pre-mRNA splicing is a key mechanism for increasing proteomic diversity and modulating gene expression. Emerging evidence indicates that splicing program is frequently deregulated during tumorigenesis, and cancer cells favor to produce protein isoforms that can promote growth and survival. Lung cancer is one of the most common cancers and the leading cause of cancer-related death worldwide. Although a number of lung cancer-related splicing events have been detected in several genome-wide analyses, much less is known about how aberrant splicing takes place in lung cancer and how it contributes to tumor development. In this study, we characterized the RNA-binding protein QKI as a new critical regulator of alternative splicing in lung cancer and as a potential marker for prognosis. Genome-wide analysis of QKI-dependent splicing by RNA-Seq identified some cancer-associated splicing changes as its targets. Our results demonstrate that QKI-5 inhibits cancer cell proliferation and prevents inappropriate activation of the Notch signaling pathway by regulating its key target, NUMB. We further showed that QKI-5 represses the inclusion of NUMB alternative exon through competing with a core splicing factor SF1. In summary, our data indicate that down-regulation of QKI causes aberrant splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated repression of Notch signaling.
Germline mutations are responsible for familial cancer syndromes which account for approximately 5–10% of all types of cancers. These mutations mainly occur at tumor suppressor genes or genome stability genes, such as DNA repair genes. Here we have identified a cancer predisposition family, in which eight members were inflicted with a wide spectrum of cancer including one diagnosed with lung cancer at 22 years old. Sequencing analysis of tumor samples as well as histologically normal specimens identified two germline mutations co-existing in the familial cancer syndrome, the mutation of tumor suppressor gene P53 V157D and mismatch repair gene PMS2 R20Q. We further demonstrate that P53 V157D and/or PMS2 R20Q mutant promotes lung cancer cell proliferation. These two mutants are capable of promoting colony formation in soft agar as well as tumor formation in transgenic drosophila system. Collectively, these data have uncovered the important role of co-existing germline P53 and PMS2 mutations in the familial cancer syndrome development.
P53 V157D; PMS2 R20Q; Germline mutation; Familial cancer syndrome; Co-existing
In addition to the previous investigations of bioactivity of aqueous extract of the edible Gracilaria tenuistipitata (AEGT) against H2O2-induced DNA damage and hepatitis C virus replication, the purpose of this study is to evaluate the potential therapeutic properties of AEGT against inflammation and hepatotoxicity using lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 cells, primary rat peritoneal macrophages and carbon tetrachloride (CCl4)-induced acute hepatitis model in rats. AEGT concentration-dependently inhibited the elevated RNA and protein levels of inducible nitric oxide synthase and cyclooxygenase-2, thereby reducing nitric oxide and prostaglandin E2 levels, respectively. Moreover, AEGT significantly suppressed the production of LPS-induced proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. These inhibitory effects were associated with the suppression of nuclear factor-kappa B activation and mitogen-activated protein kinase phosphorylation by AEGT in LPS-stimulated cells. In addition, we highlighted the hepatoprotective and curative effects of AEGT in a rat model of CCl4-intoxicated acute liver injury, which was evident from reduction in the elevated serum aspartate aminotransferase and alanine aminotransferase levels as well as amelioration of histological damage by pre-treatment or post-treatment of AEGT. In conclusion, the results demonstrate that AEGT may serve as a potential supplement in the prevention or amelioration of inflammatory diseases.
Post-translational modification (PTM) of transcriptional factors and chromatin remodelling proteins is recognized as a major mechanism by which transcriptional regulation occurs. Chromatin immunoprecipitation (ChIP) in combination with high-throughput sequencing (ChIP-seq) is being applied as a gold standard when studying the genome-wide binding sites of transcription factor (TFs). This has greatly improved our understanding of protein-DNA interactions on a genomic-wide scale. However, current ChIP-seq peak calling tools are not sufficiently sensitive and are unable to simultaneously identify post-translational modified TFs based on ChIP-seq analysis; this is largely due to the wide-spread presence of multiple modified TFs. Using SUMO-1 modification as an example; we describe here an improved approach that allows the simultaneous identification of the particular genomic binding regions of all TFs with SUMO-1 modification.
Traditional peak calling methods are inadequate when identifying multiple TF binding sites that involve long genomic regions and therefore we designed a ChIP-seq processing pipeline for the detection of peaks via a combinatorial fusion method. Then, we annotate the peaks with known transcription factor binding sites (TFBS) using the Transfac Matrix Database (v7.0), which predicts potential SUMOylated TFs. Next, the peak calling result was further analyzed based on the promoter proximity, TFBS annotation, a literature review, and was validated by ChIP-real-time quantitative PCR (qPCR) and ChIP-reChIP real-time qPCR. The results show clearly that SUMOylated TFs are able to be pinpointed using our pipeline.
A methodology is presented that analyzes SUMO-1 ChIP-seq patterns and predicts related TFs. Our analysis uses three peak calling tools. The fusion of these different tools increases the precision of the peak calling results. TFBS annotation method is able to predict potential SUMOylated TFs. Here, we offer a new approach that enhances ChIP-seq data analysis and allows the identification of multiple SUMOylated TF binding sites simultaneously, which can then be utilized for other functional PTM binding site prediction in future.
The methanolic extract of Flemingia macrophylla roots exhibited significant estrogenic activity in the transgenic plant assay system which was comparable to the activity of soybean extract. Utilizing estrogenic activity-guided fractionation, one new compound, fleminigin, together with 23 known compounds were isolated from F. macrophylla roots’ methanolic extract. The structure of the new compound was identified based on intensive spectroscopic analysis and the full spectral data for one of the isolated compounds, flemichin E, was introduced for the first time in the current investigation. The estrogenic and anti-estrogenic activities of the isolated compounds were evaluated revealing that the isolated isoflavonoids may act as partial estrogen agonists, as well as antagonists. Additionally, the anti-inflammatory and the cytotoxic activities of the isolated compounds were studied. These results suggested the potential applications of F. macrophylla extract and its isolated compounds as selective estrogen receptor modulators (SERMs).
Flemingia macrophylla; menopausal; phytoestrogen; fleminigin; flemichin E; pER8:GUS
Offspring of pregnancy complicated with preeclampsia are at high risk for hypertension, stroke and possibly obesity. The mechanisms behind the association of intrauterine exposure to preeclampsia and high risk of health problems in the later life remain largely unknown. The aims of the current investigation were to determine the changes in DNA methylation at IGF2 and GNAS DMR in offspring of preeclamptic pregnancy and to explore the possible mechanisms underlying the association between maternal preeclampsia and high risk for health problems in the later life of their offspring.
Umbilical cord blood was taken from infants born to women of preeclampsia (n=56), gestational hypertension (n=23) and normal pregnancy (n=81). DNA methylation levels of IGF2 and GNAS DMR were determined by Massarray quantitative methylation analysis. Methylation levels at IGF2 DMR were significantly lower in preeclampsia than normal pregnancy. The average methylation level at IGF2 DMR was significantly correlated with preeclampsia even after birth weight, maternal age, gestational age at delivery and fetal gender were adjusted. The difference in methylation level was not significantly different between mild and severe preeclampsia. The methylation level at GNAS DMR was not significantly correlated with birth weight, maternal age, gestational age at delivery, fetal gender, preeclampsia or gestational hypertension.
We concluded preeclampsia induced a decrease in methylation level at IGF 2 DMR, and this might be among the mechanisms behind the association between intrauterine exposure to preeclampsia and high risk for metabolic diseases in the later life of the infants.
Preeclampsia; Offspring; DNA methylation; IGF2; GNAS; Metabolic diseases
For their various bioactivities, biomaterials derived from marine algae are important ingredients in many products, such as cosmetics and drugs for treating cancer and other diseases. This mini-review comprehensively compares the bioactivities and biological functions of biomaterials from red, green, brown, and blue-green algae. The anti-oxidative effects and bioactivities of several different crude extracts of algae have been evaluated both in vitro and in vivo. Natural products derived from marine algae protect cells by modulating the effects of oxidative stress. Because oxidative stress plays important roles in inflammatory reactions and in carcinogenesis, marine algal natural products have potential for use in anti-cancer and anti-inflammatory drugs.
Algae; ROS; Antioxidant; Inflammation; Antinociceptive; Anti-cancer
Most chemotherapeutic drugs for killing cancer cells are highly cytotoxic in normal cells, which limits their clinical applications. Therefore, a continuing challenge is identifying a drug that is hypersensitive to cancer cells but has minimal deleterious effects on healthy cells. The aims of this study were to evaluate the potential of 4β-hydroxywithanolide (4βHWE) for selectively killing cancer cells and to elucidate its related mechanisms.
Methodology and Principal Findings
Changes in survival, oxidative stress, DNA damage, and apoptosis signaling were compared between 4βHWE-treated oral cancer (Ca9-22) and normal fibroblast (HGF-1) cells. At 24 h and 48 h, the numbers of Ca9-22 cells were substantially decreased, but the numbers of HGF-1 cells were only slightly decreased. Additionally, the IC50 values for 4βHWE in the Ca9-22 cells were 3.6 and 1.9 µg/ml at 24 and 48 h, respectively. Time-dependent abnormal increases in ROS and dose-responsive mitochondrial depolarization can be exploited by using 4βHWE in chemotherapies for selectively killing cancer cells. Dose-dependent DNA damage measured by comet-nuclear extract assay and flow cytometry-based γ-H2AX/propidium iodide (PI) analysis showed relatively severer damage in the Ca9-22 cells. At both low and high concentrations, 4βHWE preferably perturbed the cell cycle in Ca9-22 cells by increasing the subG1 population and arrest of G1 or G2/M. Selective induction of apoptosis in Ca9-22 cells was further confirmed by Annexin V/PI assay, by preferential expression of phosphorylated ataxia-telangiectasia- and Rad3-related protein (p-ATR), and by cleavage of caspase 9, caspase 3, and poly ADP-ribose polymerase (PARP).
Together, the findings of this study, particularly the improved understanding of the selective killing mechanisms of 4βHWE, can be used to improve efficiency in killing oral cancer cells during chemoprevention and therapy.
Liriope spicata is a well-known herb in traditional Chinese medicine, and its root has been clinically demonstrated to be effective in the treatment of metabolic and neural disorders. The constituents isolated from Liriope have also recently been shown to possess anticancer activity, although the mechanism of which remains largely unknown. Here, we illustrate the anticancer activity of LPRP-9, one of the active fractions we fractionated from the Liriope platyphylla root part (LPRP) extract. Treatment with LPRP-9 significantly inhibited proliferation of cancer cell lines MCF-7 and Huh-7 and down-regulated the phosphorylation of AKT. LPRP-9 also activates the stress-activated MPAK, JNK, p38 pathways, the p53 cell-cycle checkpoint pathway, and a series of caspase cascades while downregulating expression of antiapoptotic factors Bcl-2, Bcl-XL, and survivin. Such activities strongly suggest a role for LPRP-9 in apoptosis and autophagy. We further purified and identified the compound (−)-Liriopein B from LPRP-9, which is capable of inhibiting AKT phosphorylation at low concentration. The overall result highlights the anticancer property of LPRP-9, suggests its mechanism for inhibition of proliferation and promotion of cell death for cancer cells via regulation of multitarget pathways, and denotes the importance of purifying components of fraction LPRP-9 to aid cancer therapy.
Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase gene have recently been described in a subset of non–small-cell lung cancers (NSCLCs). Because little is known about these tumors, we examined the clinical characteristics and treatment outcomes of patients with NSCLC with ROS1 rearrangement.
Patients and Methods
Using a ROS1 fluorescent in situ hybridization (FISH) assay, we screened 1,073 patients with NSCLC and correlated ROS1 rearrangement status with clinical characteristics, overall survival, and when available, ALK rearrangement status. In vitro studies assessed the responsiveness of cells with ROS1 rearrangement to the tyrosine kinase inhibitor crizotinib. The clinical response of one patient with ROS1-rearranged NSCLC to crizotinib was investigated as part of an expanded phase I cohort.
Of 1,073 tumors screened, 18 (1.7%) were ROS1 rearranged by FISH, and 31 (2.9%) were ALK rearranged. Compared with the ROS1-negative group, patients with ROS1 rearrangements were significantly younger and more likely to be never-smokers (each P < .001). All of the ROS1-positive tumors were adenocarcinomas, with a tendency toward higher grade. ROS1-positive and -negative groups showed no difference in overall survival. The HCC78 ROS1-rearranged NSCLC cell line and 293 cells transfected with CD74-ROS1 showed evidence of sensitivity to crizotinib. The patient treated with crizotinib showed tumor shrinkage, with a near complete response.
ROS1 rearrangement defines a molecular subset of NSCLC with distinct clinical characteristics that are similar to those observed in patients with ALK-rearranged NSCLC. Crizotinib shows in vitro activity and early evidence of clinical activity in ROS1-rearranged NSCLC.
This article provides a fully Bayesian approach for modeling of single-dose and complete pharmacokinetic data in a population pharmacokinetic (PK) model. To overcome the impact of outliers and the difficulty of computation, a generalized linear model is chosen with the hypothesis that the errors follow a multivariate Student t distribution which is a heavy-tailed distribution. The aim of this study is to investigate and implement the performance of the multivariate t distribution to analyze population pharmacokinetic data. Bayesian predictive inferences and the Metropolis-Hastings algorithm schemes are used to process the intractable posterior integration. The precision and accuracy of the proposed model are illustrated by the simulating data and a real example of theophylline data.
Hepatitis C virus (HCV) is an important human pathogen leading to hepatocellular carcinoma. Using an in vitro cell-based HCV replicon and JFH-1 infection system, we demonstrated that an aqueous extract of the seaweed Gracilaria tenuistipitata (AEGT) concentration-dependently inhibited HCV replication at nontoxic concentrations. AEGT synergistically enhanced interferon-α (IFN-α) anti-HCV activity in a combination treatment. We found that AEGT also significantly suppressed virus-induced cyclooxygenase-2 (COX-2) expression at promoter transactivation and protein levels. Notably, addition of exogenous COX-2 expression in AEGT-treated HCV replicon cells gradually abolished AEGT anti-HCV activity, suggesting that COX-2 down-regulation was responsible for AEGT antiviral effects. Furthermore, we highlighted the inhibitory effect of AEGT in HCV-induced pro-inflammatory gene expression such as the expression of tumour necrosis factor-α, interleukin-1β, inducible nitrite oxide synthase and COX-2 in a concentration-dependent manner to evaluate the potential therapeutic supplement in the management of patients with chronic HCV infections.
The opportunistic enterobacterium, Morganella morganii, which can cause bacteraemia, is the ninth most prevalent cause of clinical infections in patients at Changhua Christian Hospital, Taiwan. The KT strain of M. morganii was isolated during postoperative care of a cancer patient with a gallbladder stone who developed sepsis caused by bacteraemia. M. morganii is sometimes encountered in nosocomial settings and has been causally linked to catheter-associated bacteriuria, complex infections of the urinary and/or hepatobiliary tracts, wound infection, and septicaemia. M. morganii infection is associated with a high mortality rate, although most patients respond well to appropriate antibiotic therapy. To obtain insights into the genome biology of M. morganii and the mechanisms underlying its pathogenicity, we used Illumina technology to sequence the genome of the KT strain and compared its sequence with the genome sequences of related bacteria.
The 3,826,919-bp sequence contained in 58 contigs has a GC content of 51.15% and includes 3,565 protein-coding sequences, 72 tRNA genes, and 10 rRNA genes. The pathogenicity-related genes encode determinants of drug resistance, fimbrial adhesins, an IgA protease, haemolysins, ureases, and insecticidal and apoptotic toxins as well as proteins found in flagellae, the iron acquisition system, a type-3 secretion system (T3SS), and several two-component systems. Comparison with 14 genome sequences from other members of Enterobacteriaceae revealed different degrees of similarity to several systems found in M. morganii. The most striking similarities were found in the IS4 family of transposases, insecticidal toxins, T3SS components, and proteins required for ethanolamine use (eut operon) and cobalamin (vitamin B12) biosynthesis. The eut operon and the gene cluster for cobalamin biosynthesis are not present in the other Proteeae genomes analysed. Moreover, organisation of the 19 genes of the eut operon differs from that found in the other non-Proteeae enterobacterial genomes.
This is the first genome sequence of M. morganii, which is a clinically relevant pathogen. Comparative genome analysis revealed several pathogenicity-related genes and novel genes not found in the genomes of other members of Proteeae. Thus, the genome sequence of M. morganii provides important information concerning virulence and determinants of fitness in this pathogen.
Our study aimed to investigate the prevalence of cognitive impairment(CI) and the associated risk factors among elderly people in Shanghai urban area, China.
A population-based survey was conducted among people aged 55 years or older in urban areas of Shanghai. Face-to-face interviews were carried out to collect information including demographic characteristics, medical history, and medication use, etc. The validated Chinese version of the Mini-Mental State Examination(MMSE) was used to screen subjects with CI, and the criteria of CI were adjusted for education levels.
A total of 3,176 home-living residents (≥55 years old) were included in the study. Among them, 266 people (102 men and 164 women) were identified as cognition impaired, with a prevalence of 8.38% (266/3,176, 95% CI: (8.26, 8.49)) for both genders, 9.21% (102/1,107,95% CI: (9.18, 9.33)) for men and 7.93% (164/2,069, 95% CI: (7.80, 8.09)) for women, respectively. Furthermore, we found that several significant risk factors, including social factors(education, number of children, marriage status, and family structure), physiological factors (age, blood glucose level, and obesity), factors on living styles(physical exercise, diet & chronic diseases), and genetic factor(ApoE), associated with CI onset.
This study confirms the high prevalence of CI among the elderly population in the Shanghai urban in China, similar to previous epidemiologic studies in Western countries. The putative risk factors associated with CI merit further investigated.
Cognitive impairment; Prevalence; Risk factors
Methanolic extracts of Gracilaria tenuistipitata (MEGT) were obtained from the edible red algae. Previously, we found that water extract of G. tenuistipitata was able to modulate oxidative stress-induced DNA damage and its related cellular responses.
In this study, the methanol extraction product MEGT was used to evaluate the cell growth inhibition in oral cancer cells and its possible mechanism was investigated.
The cell viability of MEGT treated Ca9-22 oral cancer cell line was significantly decreased in a dose–response manner (p < 0.05). The sub-G1 population and annexin V intensity of MEGT-treated Ca9-22 cancer cells were significantly increased in a dose–response manner (p < 0.0005 and p < 0.001, respectively). The γH2AX intensities of MEGT-treated Ca9-22 cancer cells were significantly increased in a dose–response manner (p < 0.05). The reactive oxygen species (ROS) and glutathione (GSH)-positive intensities of MEGT-treated Ca9-22 oral cancer cells were significantly increased and decreased, respectively, in a dose–response manner (p < 0.05). The DiOC2(3) intensity for mitochondrial membrane potential (MMP) of MEGT-treated Ca9-22 cancer cells was significantly decreased in a dose–response manner (p < 0.05).
These results indicated that MEGT had apoptosis-based cytotoxicity against oral cancer cells through the DNA damage, ROS induction, and mitochondrial depolarization. Therefore, MEGT derived from the edible algae may have potential therapeutic effects against oral squamous cell carcinoma (OSCC).
Red algae; Oral cancer; Apoptosis; γ-H2AX; ROS; Mitochondrial membrane potential; Glutathione
The binding of IL-18 to IL-18Rα induces both pro-inflammatory and protective functions during infection, depending on the context in which it occurs. IL-18 is highly expressed in the liver of wild type (WT) C57BL/6 mice following lethal infection with highly virulent Ixodes Ovatus Ehrlichia (IOE), an obligate intracellular bacterium that causes acute fatal toxic shock-like syndrome. In this study, we found that IOE infection of IL-18Rα-/- mice resulted in significantly less host cell apoptosis, decreased hepatic leukocyte recruitment, enhanced bacterial clearance and prolonged survival compared to infected WT mice, suggesting a pathogenic role of IL-18/IL-18Rα in Ehrlichia-induced toxic shock. Although lack of IL-18R decreases the magnitude of IFN-γ producing type-1 immune response, enhanced resistance of the IL-18Rα-/- mice against Ehrlichia correlated with increased pro-inflammatory cytokines at sites of infection, decreased systemic IL-10 production, increased frequency of protective natural killer T (NKT) cells producing TNF-α and IFN-γ and decreased frequency of pathogenic TNF-α-producing CD8+ T cells. Adoptive transfer of immune wild type CD8+ T cells increased bacterial burden in IL-18Rα-/- mice following IOE infection. Furthermore, rIL-18 treatment of WT mice infected with mildly virulent Ehrlichia muris (EM) impaired bacterial clearance and enhanced liver injury. Finally, lack of IL-18R signal reduced dendritic cells (DCs) maturation and their TNF-α production, suggesting that IL-18 possibly promote the adaptive pathogenic immune responses against Ehrlichia via influencing T cell priming functions of DCs Together, these results suggest that the presence or absence of IL-18R signals governs the pathogenic versus protective immunity in a model of Ehrlichia-induced immunopathology.
IL-18; IL-18 receptor alpha; Ehrlichiosis
RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. The most frequently cloned satsiRNA, satsiR-12, was predicted to imperfectly match to Cucumber mosaic virus (CMV) RNAs in the upstream area of the 3′ untranslated region (3′ UTR). Moreover, an artificial satsiR-12 (asatsiR-12) mediated cleavage of a green fluorescent protein (GFP) sensor construct harboring the satsiR-12 target site. asatsiR-12 also mediated reduction of viral RNAs in 2b-deficient CMV (CMVΔ2b)-infected Nicotiana benthamiana. The reduction was not observed in CMVΔ2b-infected RDR6i plants, in which RDR6 was silenced. Following infection with 2b-containing CMV, the reduction in viral RNAs was not observed in plants of either genotype, indicating that the asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b protein. Our results suggest that satsiR-12 targeting the 3′ UTR of CMV RNAs triggered RDR6-dependent antiviral silencing. Competition experiments with wild-type CMV RNAs and anti-satsiR-12 mutant RNA1 in the presence of 2b and satRNA demonstrate the inhibitory effect of the 2b protein on the satsiR-12-related degradation of CMV RNAs, revealing a substantial suppressor function of the 2b protein in native CMV infection. Our data provide evidence for the important biological functions of satsiRNAs in homeostatic interactions among the host, virus, and satRNA in the final outcome of viral infection.
Withanolides are a large group of steroidal lactones found in Solanaceae plants that exhibit potential anticancer activities. We have previously demonstrated that a withanolide, tubocapsenolide A, induced cycle arrest and apoptosis in human breast cancer cells, which was associated with the inhibition of heat shock protein 90 (Hsp90). To investigate whether other withanolides are also capable of inhibiting Hsp90 and to analyze the structure-activity relationships, nine withanolides with different structural properties were tested in human breast cancer cells MDA-MB-231 and MCF-7 in the present study. Our data show that the 2,3-unsaturated double bond-containing withanolides inhibited Hsp90 function, as evidenced by selective depletion of Hsp90 client proteins and induction of Hsp70. The inhibitory effect of the withanolides on Hsp90 chaperone activity was further confirmed using in vivo heat shock luciferase activity recovery assays. Importantly, Hsp90 inhibition by the withanolides was correlated with their ability to induce cancer cell death. In addition, the withanolides reduced constitutive NF-κB activation by depleting IκB kinase complex (IKK) through inhibition of Hsp90. In estrogen receptor (ER)-positive MCF-7 cells, the withanolides also reduced the expression of ER, and this may be partly due to Hsp90 inhibition. Taken together, our results suggest that Hsp90 inhibition is a general feature of cytotoxic withanolides and plays an important role in their anticancer activity.
In a structure-activity relationship (SAR) study, 3-methoxy-1,4-phenanthrenequinones, calanquinone A (6a), denbinobin (6b), 5-OAc-calanquinone A (7a) and 5-OAc-denbinobin (7b), have significantly promising cytotoxicity against various human cancer cell lines (IC50 0.08–1.66 µg/mL). Moreover, we also established a superior pharmacophore model for cytotoxicity (r = 0.931) containing three hydrogen bond acceptors (HBA1, HBA2 and HBA3) and one hydrophobic feature (HYD) against MCF-7 breast cancer cell line. The pharmacophore model indicates that HBA3 is an essential feature for the oxygen atom of 5-OH in 6a–b and for the carbonyl group of 5-OCOCH3 in 7a–b, important for their cytotoxic properties. The SAR for moderately active 5a–b (5-OCH3), and highly active 6a–b and 7a–b, are also elaborated in a spatial aspect model. Further rational design and synthesis of new cytotoxic phenanthrene analogs can be implemented via this model. Additionally, employing a ChemGPS-NP based model for cytotoxicity mode of action (MOA) provides support for a preliminary classification of compounds 6a–b as topoisomerase II inhibitors.
Due to its strong immune stimulatory effects through TLR9, CpG-containing oligodeoxynucleotides (CpG ODN) have been tested in multiple clinical trials as vaccine adjuvant for infectious diseases and cancer. However, immune suppression induced by systemic administration of CpGs has been reported recently. In this study, we evaluated the impact of CpGs in an acute rickettsiosis model. We found that systemic treatment with type B CpG (CpG-B), but not type A CpG (CpG-A), at 2 days after sublethal R. australis infection induced mouse death. Although wild-type (WT) B6 and IDO−/− mice showed similar survival rates with three different doses of R. australis infection, treatment with CpG-B after sublethal infection consistently induced higher mortality with greater tissue bacterial loads in WT but not IDO−/− mice. Also, CpG-B treatment promoted the development of higher serum concentrations of proinflammatory cytokines/chemokines through IDO. Furthermore, while T cell-mediated immune responses enhanced by CpG-B were independent of IDO, treatment with CpG-B promoted T cell activation, PD-1 expression and cell apoptosis partially through IDO. A depletion study using anti-mPDCA-1 mAb indicated that plasmacytoid dendritic cells (pDC) were not required for CpG-B-induced death of R. australis-infected mice. Additionally, the results in iNOS−/− mice suggested that nitric oxide (NO) was partially involved in CpG-B-induced death of R. australis-infected mice. Surprisingly, pre-treatment with CpG-B before administration of a lethal dose of R. australis provided effective immunity in WT, IDO−/− and iNOS−/− mice. Taken together, our study provides evidence that CpGs exert complex immunological effects by both IDO-dependent and -independent mechanisms, and that systemic treatment with CpGs before or after infection has a significant and distinct impact on disease outcomes.