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1.  Apoptosis of U937 Cells Induced by Hematoporphyrin Monomethyl Ether-Mediated Sonodynamic Action 
The present study aims to investigate apoptosis of U937 cells induced by hematoporphyrin monomethyl ether (HMME)-mediated sonodynamic therapy (SDT).
HMME concentration was kept constant at 10 μg/mL. Tumor cells suspended in serum-free RPM1640 were exposed to ultrasound at 1.1 MHz for up to 60 seconds with an intensity of 1 W/cm2 in the presence and absence of HMME. The viability of cells was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT) test. Apoptosis was analyzed using a flow cytometer with Annexin V-PE/7-ADD staining as well as fluorescence microscopy with 4′-6-diamidino-2-phenylindole (DAPI) staining. The DNA damage of U937 cells, intracellular reactive oxygen species (ROS), and mitochondria membrane potential (MMP) were also analyzed by a flow cytometer after exposures. Western blotting and reverse transcriptase–polymerase chain reaction were used to analyze the protein and mRNA expression level of caspase-3 and poly(ADP-ribose) polymerase (PARP).
Fluorescent imaging revealed that HMME mainly localized in the mitochondria. MTT assay showed 55.6% of cell survival at 4 hours post-SDT. Flow cytometric analysis displayed a significant increase in the early- and late-apoptotic cell populations (35.6%) of U937 cells by HMME-mediated SDT. Compared with the control, ultrasound-alone, and HMME-alone groups, the intracellular ROS and the MMP loss were greatly increased in the combined SDT group. Obvious nuclear condensation was also found with DAPI staining, and the DNA fragment increased to 33.9% at 2 hours post-SDT treatment. Immunofluorescent staining indicated obvious Bax translocation after SDT. Western blot showed visible enhancement of caspase-3 and PARP cleavage. In addition, caspase-3 and PARP mRNA expression of U937 cells increased remarkably after SDT treatment.
The findings demonstrated that HMME-mediated sonodynamic action (HMME-SDT) significantly induced apoptosis of U937 cells, suggesting that HMME may be a good sonosensitizer, and HMME-SDT might be a potential therapeutic strategy for cancer treatment.
PMCID: PMC3615696  PMID: 23506428
apoptosis; HMME; leukemia U937 cells; sonodynamic therapy
2.  Increased peripheral and local soluble FGL2 in the recovery of renal ischemia reperfusion injury in a porcine kidney auto-transplantation model 
Regulatory T cells (Treg) protect kidney against ischemia reperfusion (IR) injury via suppressing innate immunity, but the mechanism has not been fully clarified. Soluble fibrinogen-like protein 2 (sFGL2), a novel effector of Treg, may affect apoptosis and inflammation. This study investigated the role of sFGL2 in renal IR injury in a porcine kidney auto-transplantation model.
Materials and methods
The left kidney was retrieved from mini pigs and infused by University of Wisconsin solution into the renal artery with the renal artery and vein clamped for 24-h cold storage. After the right nephrectomy, the left kidney was auto-transplanted into the right for 2 weeks. 3 pigs were sacrificed at day 2, 5, 7, 10 and 14 post-transplantation respectively. Collected renal tissues and daily blood samples were stored for further analyses.
Both serum creatinine and blood urea nitrogen were maximized during day 2 to 5 and followed by a gradual recovery over 2 weeks. The similar pattern were showed in histological damage, myeloperoxidase + cells and apoptosis in the kidney, as well as circulating TNF-α and IFN-γ. Serum sFGL2 presented a fluctuating increase and reached a peak at day 10. The expression of sFGL2 and its receptor FcγRIIB as well as Foxp3 and IL-10 in the kidney was notably increased from day 5 to 10.
The increased sFGL2 together with FcγRIIB during renal recovery after IR injury suggested that sFGL2 might be a potential renoprotective mediator involved in the renal self-repairing and remodeling in this 2-week porcine auto-transplantation model.
PMCID: PMC3936847  PMID: 24559374
Soluble FGL2; Ischemia reperfusion injury; Kidney auto-transplantation; Porcine; FcγRIIB
3.  Obstruction of Dengue Virus Maturation by Fab Fragments of the 2H2 Antibody 
Journal of Virology  2013;87(16):8909-8915.
The 2H2 monoclonal antibody recognizes the precursor peptide on immature dengue virus and might therefore be a useful tool for investigating the conformational change that occurs when the immature virus enters an acidic environment. During dengue virus maturation, spiky, immature, noninfectious virions change their structure to form smooth-surfaced particles in the slightly acidic environment of the trans-Golgi network, thereby allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions become fully infectious when they release the cleaved precursor peptide upon reaching the neutral-pH environment of the extracellular space. Here we report on the cryo-electron microscopy structures of the immature virus complexed with the 2H2 antigen binding fragments (Fab) at different concentrations and under various pH conditions. At neutral pH and a high concentration of Fab molecules, three Fab molecules bind to three precursor-membrane proteins on each spike of the immature virus. However, at a low concentration of Fab molecules and pH 7.0, only two Fab molecules bind to each spike. Changing to a slightly acidic pH caused no detectable change of structure for the sample with a high Fab concentration but caused severe structural damage to the low-concentration sample. Therefore, the 2H2 Fab inhibits the maturation process of immature dengue virus when Fab molecules are present at a high concentration, because the three Fab molecules on each spike hold the precursor-membrane molecules together, thereby inhibiting the normal conformational change that occurs during maturation.
PMCID: PMC3754034  PMID: 23740974
4.  Selective intra-arterial infusion of rAd-p53 with chemotherapy for advanced oral cancer: a randomized clinical trial 
BMC Medicine  2014;12:16.
In this study, a combination of recombinant adenoviral p53 (rAd-p53) gene therapy and intra-arterial delivery of chemotherapeutic agents for treatment of oral squamous cell carcinoma was evaluated.
In total, 99 patients with stage III or IV oral carcinoma who had refused or were ineligible for surgery were enrolled in a randomized, placebo-controlled, double-blind, phase III clinical trial. They were randomly assigned to group I (n = 35; intra-arterial infusion of rAd-p53 plus chemotherapy), group II (n = 33; intra-arterial infusion of rAd-p53 plus placebo chemotherapy), or group III (n = 31; intra-arterial infusion of placebo rAd-p53 plus chemotherapy).
The median length of follow-up was 36 months (range, 3 to 86 months). During follow-up, 16 patients in group I, 20 in group II, and 22 in group III died. Group I (48.5%) had a higher complete response rate than groups II (16.7%) and III (17.2%) (P = 0.006). The rate of non-responders in group I was significantly lower than that in groups II and III (P < 0.020). A log-rank test for survival rate indicated that group I had a significantly higher survival rate than group III (P = 0.019). The survival rate of patients with stage III but not stage IV oral cancer was significantly higher in group I than in group III (P = 0.015, P = 0.200, respectively). The survival rate of patients with stage IV did not differ significantly among the three groups. Or the 99 patients, 63 patients experienced adverse events of either transient flu-like symptoms or bone marrow suppression, while 13 patients had both these conditions together. No replication-deficient virus was detected in patient serum, urine, or sputum. rAd-p53 treatment increased Bax expression in the primary tumor of 80% of patients, as shown by immunohistochemical staining.
Intra-arterial infusion of combined rAd-p53 and chemotherapy significantly increased the survival rate of patients with stage III but not stage IV oral cancer, compared with intra-arterial chemotherapy. Intra-arterial infusion of combined rAd-p53 and chemotherapy may represent a promising alternative treatment for oral squamous cell carcinoma.
Trial registration
ChiCTR-TRC-09000392 (Date of registration: 2009-05-18).
PMCID: PMC3922639  PMID: 24479409
Oral carcinoma; Gene therapy; Chemotherapy; Intra-arterial infusion; p53
5.  Atheroprotective Effect of Oleoylethanolamide (OEA) Targeting Oxidized LDL 
PLoS ONE  2014;9(1):e85337.
Dietary fat-derived lipid oleoylethanolamide (OEA) has shown to modulate lipid metabolism through a peroxisome proliferator-activated receptor-alpha (PPAR-α)-mediated mechanism. In our study, we further demonstrated that OEA, as an atheroprotective agent, modulated the atherosclerotic plaques development. In vitro studies showed that OEA antagonized oxidized LDL (ox-LDL)-induced vascular endothelial cell proliferation and vascular smooth muscle cell migration, and suppressed lipopolysaccharide (LPS)-induced LDL modification and inflammation. In vivo studies, atherosclerosis animals were established using balloon-aortic denudation (BAD) rats and ApoE-/- mice fed with high-caloric diet (HCD) for 17 or 14 weeks respectively, and atherosclerotic plaques were evaluated by oil red staining. The administration of OEA (5 mg/kg/day, intraperitoneal injection, i.p.) prevented or attenuated the formation of atherosclerotic plaques in HCD-BAD rats or HCD-ApoE−/− mice. Gene expression analysis of vessel tissues from these animals showed that OEA induced the mRNA expressions of PPAR-α and downregulated the expression of M-CFS, an atherosclerotic marker, and genes involved in oxidation and inflammation, including iNOS, COX-2, TNF-α and IL-6. Collectively, our results suggested that OEA exerted a pharmacological effect on modulating atherosclerotic plaque formation through the inhibition of LDL modification in vascular system and therefore be a potential candidate for anti-atherosclerosis drug.
PMCID: PMC3896367  PMID: 24465540
6.  The protective effect of baicalin against renal ischemia-reperfusion injury through inhibition of inflammation and apoptosis 
Renal ischemia-reperfusion injury (IRI) increases the rates of acute kidney failure, delayed graft function, and early mortality after kidney transplantation. The pathophysiology involved includes oxidative stress, mitochondrial dysfunction, and immune-mediated injury. The anti-oxidation, anti-apoptosis, and anti-inflammation properties of baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, have been verified. This study therefore assessed the effects of baicalin against renal IRI in rats.
Baicalin was intraperitoneally injected 30 min before renal ischemia. Serum and kidneys were harvested 24 h after reperfusion. Renal function and histological changes were assessed. Markers of oxidative stress, the Toll-like receptor (TLR)2 and TLR4 signaling pathway, mitochondrial stress, and cell apoptosis were also evaluated.
Baicalin treatment decreased oxidative stress and histological injury, and improved kidney function, as well as inhibiting proinflammatory responses and tubular apoptosis. Baicalin pretreatment also reduced the expression of TLR2, TLR4, MyD88, p-NF-κB, and p-IκB proteins, as well as decreasing caspase-3 activity and increasing the Bcl-2/Bax ratio.
Baicalin may attenuate renal ischemia-reperfusion injury by inhibiting proinflammatory responses and mitochondria-mediated apoptosis. These effects are associated with the TLR2/4 signaling pathway and mitochondrial stress.
PMCID: PMC3893527  PMID: 24417870
Baicalin; Ischemia-reperfusion; Kidney; Inflammation; Apoptosis
7.  Investigations of the functional states of dendritic cells under different conditioned microenvironments by Fourier transformed infrared spectroscopy 
Dendritic cells are potent and specialized antigen presenting cells, which play a crucial role in initiating and amplifying both the innate and adaptive immune responses. The dendritic cell-based vaccination against cancer has been clinically achieved promising successes. But there are still many challenges in its clinical application, especially for how to identify the functional states.
The CD14+ monocytes were isolated from human peripheral blood after plastic adherence and purified to approximately 98% with cocktail immunomagnetic beads. The immature dendritic cells and mature dendritic cells were induced by traditional protocols. The resulting dendritic cells were cocultured with normal cells and cancer cells. The functional state of dendritic cells including immature dendritic cells (imDCs) and mature dendritic cells (mDCs) under different conditioned microenvironments were investigated by Fourier transformed infrared spectroscopy (FTIR) and molecular biological methods.
The results of Fourier transformed infrared spectroscopy showed that the gene transcription activity and energy states of dendritic cells were specifically suppressed by tumor cells (P < 0.05 or 0.01). The expression levels of NF-kappa B (NF-κB) in dendritic cells were also specifically inhibited by tumor-derived factors (P < 0.05 or 0.01). Moreover, the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were closely correlated with the expression levels of NF-κB (R2:0.69 and R2:0.81, respectively).
Our results confirmed that the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were positively correlated with the expression levels of NF-κB, suggesting that Fourier transformed infrared spectroscopy technology could be clinically applied to identify the functional states of dendritic cell when performing dendritic cell-based vaccination. It’s significant for the simplification and standardization of dendritic cell-based vaccination clinical preparation protocols.
PMCID: PMC3897965  PMID: 24410930
Dendritic cell; Fourier transformed infrared spectroscopy; Functional states of cells; NF-κB
8.  Correction: Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells 
PLoS Genetics  2014;10(1):10.1371/annotation/e7c49fcc-0bc2-4aa9-bbcf-f4b386eb2cd0.
PMCID: PMC3888203
9.  Demystifying the nuclear function of Argonaute proteins 
RNA Biology  2014;11(1):18-24.
The Argonaute family of proteins is highly evolutionarily conserved and plays essential roles in small RNA-mediated gene regulatory pathways and in a wide variety of cellular processes. They were initially discovered by genetics studies in plants and have been well characterized as key components of gene silencing pathways guided by small RNAs, a phenomenon known as RNA interference. Conventionally, guided by different classes of small RNAs, Argonautes bind to and silence homologous target sequences at the post-transcriptional level. Increasing lines of evidence support their multi-functional roles in the nucleus. Advances in high-throughput genome-wide methodologies have greatly facilitated our understanding of their functions in post-transcriptional gene silencing as well as in other nuclear events. In this point-of-view, we will summarize key findings from genome-wide analyses of the Ago subfamily of proteins in mammals and Drosophila, discuss their nuclear functions in the regulation of transcription and alternative splicing identified in recent years, and briefly touch upon their potential implications in cancer.
PMCID: PMC3929419  PMID: 24384674
Argonaute proteins; histone modifications; transcription regulation; alternative splicing; cancer
10.  Prevalence of Human Papillomavirus and Cervical Intraepithelial Neoplasia in China: A pooled analysis of 17 Population-based Studies 
High-risk (HR) human papillomavirus (HPV) prevalence has been shown to correlate well with cervical cancer incidence rates. Our study aimed to estimate the prevalence of HR-HPV and cervical intraepithelial neoplasia (CIN) in China and indirectly inform on the cervical cancer burden in the country. 30,207 women from 17 population-based studies throughout China were included. All women received HPV DNA testing (HC2, Qiagen), visual inspection with acetic acid, and liquid-based cytology. Women positive for any test received colposcopy-directed or 4-quadrant biopsies. 29,579 women had HR-HPV testing results, of whom 28,761 had biopsy-confirmed (9019, 31.4%) or assumed (19,742, 68.6%) final diagnosis. Overall crude HR-HPV prevalence was 17.7%. HR-HPV prevalence was similar in rural and urban areas but showed dips in different age groups: at age 25–29 years (11.3%) in rural and at age 35–39 (11.3%) in urban women. In rural and urban women, age-standardized CIN2 prevalence was 1.5% (95%CI: 1.4%–1.6%) and 0.7% (95%CI: 0.7%–0.8%), and CIN3+ prevalence was 1.2% (95%CI: 1.2%–1.3%) and 0.6% (95%CI: 0.5%–0.7%), respectively. Prevalence of CIN3+ as a percentage of either all women or HR-HPV positive women steadily increased with age, peaking in 45–49 year-old women. High prevalence of HR-HPV and CIN3+ was detected in both rural and urban China. The steady rise of CIN3+ up to the age group 45–49 is attributable to lack of lesion removal through screening. Our findings document the inadequacy of current screening in China while indirectly raising the possibility that the cervical cancer burden in China is under-reported.
PMCID: PMC3435460  PMID: 22488743
Cervical Cancer; Human Papillomavirus; Cervical Intraepithelial Neoplasia; Prevalence; China
11.  Gut-lung crosstalk in pulmonary involvement with inflammatory bowel diseases 
Pulmonary abnormalities, dysfunction or hyper-reactivity occurs in association with inflammatory bowel disease (IBD) more frequently than previously recognized. Emerging evidence suggests that subtle inflammation exists in the airways among IBD patients even in the absence of any bronchopulmonary symptoms, and with normal pulmonary functions. The pulmonary impairment is more pronounced in IBD patients with active disease than in those in remission. A growing number of case reports show that the IBD patients develop rapidly progressive respiratory symptoms after colectomy, with failure to isolate bacterial pathogens on repeated sputum culture, and often request oral corticosteroid therapy. All the above evidence indicates that the inflammatory changes in both the intestine and lung during IBD. Clinical or subclinical pulmonary inflammation accompanies the main inflammation of the bowel. Although there are clinical and epidemiological reports of chronic inflammation of the pulmonary and intestinal mucosa in IBD, the detailed mechanisms of pulmonary-intestinal crosstalk remain unknown. The lung has no anatomical connection with the main inflammatory site of the bowel. Why does the inflammatory process shift from the gastrointestinal tract to the airways? The clinical and subclinical pulmonary abnormalities, dysfunction, or hyper-reactivity among IBD patients need further evaluation. Here, we give an overview of the concordance between chronic inflammatory reactions in the airways and the gastrointestinal tract. A better understanding of the possible mechanism of the crosstalk among the distant organs will be beneficial in identifying therapeutic strategies for mucosal inflammatory diseases such as IBD and allergy.
PMCID: PMC3812478  PMID: 24187454
Inflammatory bowel disease; Pulmonary symptoms; Gut-lung crosstalk; Biao-Li relationship; Social manner
12.  Chromatin remodeling by the small RNA machinery in mammalian cells 
Epigenetics  2013;9(1):45-52.
Chromatin states, quite different from changes in DNA sequence, can impact fundamental cellular processes such as determination of cell identity and development of disease. However, how chromatin states are established and regulated remain to be fully elucidated. In several lower eukaryotes, the small RNA machinery comprised of small RNA and its partners, the Argonaute proteins, is known to play important roles in the establishment of heterochromatin and silencing of repetitive sequences. In mammalian cells, however, the nuclear function of the small RNA machinery is largely unknown. Emerging evidence suggests that components of the small RNA pathway interact with chromatin to regulate nuclear events, including gene transcription and alternative splicing. In addition, these endogenous mechanisms are being exploited to target specific genomic loci for manipulation of gene expression and splicing events. In this review, I summarize current understanding of chromatin remodeling by small RNAs in mammalian cells and highlight recent efforts to map genome-wide interactions between RNAi-related factors and chromatin.
PMCID: PMC3928185  PMID: 24149777
small RNA; Argonaute; epigenetics; chromatin; RNA activation; miRNA; transcription
13.  Origin, Evolution, and Genotyping of Emergent Porcine Epidemic Diarrhea Virus Strains in the United States 
mBio  2013;4(5):e00737-13.
Coronaviruses are known to infect humans and other animals and cause respiratory and gastrointestinal diseases. Here we report the emergence of porcine epidemic diarrhea virus (PEDV) in the United States and determination of its origin, evolution, and genotypes based on temporal and geographical evidence. Histological lesions in small intestine sections of affected pigs and the complete genomic sequences of three emergent strains of PEDV isolated from outbreaks in Minnesota and Iowa were characterized. Genetic and phylogenetic analyses of the three U.S. strains revealed a close relationship with Chinese PEDV strains and their likely Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, which can be classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the divergent time based on the complete genomic sequences is consistent with the actual time difference, approximately 2 to 3 years, of the PED outbreaks between China (December 2010) and the United States (May 2013). The finding that the emergent U.S. PEDV strains share unique genetic features at the 5′-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission. The data from this study have important implications for understanding the ongoing PEDV outbreaks in the United States and will guide future efforts to develop effective preventive and control measures against PEDV.
The sudden emergence of porcine epidemic diarrhea virus (PEDV), a coronavirus, for the first time in the United States causes significant economic and public health concerns. Since its recognition in May 2013, PEDV has rapidly spread across the United States, resulting in high mortality in piglets in more than 17 States now. The ongoing outbreaks of Middle East respiratory syndrome coronavirus in humans from countries in or near the Arabian Peninsula and the historical deadly nature of the 2002 outbreaks of severe acute respiratory syndrome coronavirus create further anxiety over the emergence of PEDV in the United States due to the lack of scientific information about the origin and evolution of this emerging coronavirus. Here we report the detailed genetic characterization, origin, and evolution of emergent PEDV strains in the United States. The results provide much needed information to devise effective preventive and control strategies against PEDV in the United States.
PMCID: PMC3812708  PMID: 24129257
14.  Targeted p21WAF1/CIP1 Activation by RNAa Inhibits Hepatocellular Carcinoma Cells 
Nucleic Acid Therapeutics  2012;22(5):335-343.
RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double-stranded RNA (dsRNA), also known as small activating RNA (saRNA). p21WAF1/CIP1 (p21) is a putative tumor suppressor gene due to its role as a key negative regulator of the cell cycle and cell proliferation. It is frequently downregulated in cancer including hepatocellular carcinoma (HCC), but is rarely mutated or deleted, making it an ideal target for RNAa-based overexpression to restore its tumor suppressor function. In the present study, we investigated the antigrowth effects of p21 RNAa in HCC cells. Transfection of a p21 saRNA (dsP21-322) into HepG2 and Hep3B cells significantly induced the expression of p21 at both the mRNA and protein levels, and inhibited cell proliferation and survival. Further analysis of dsP21-322 transfected cells revealed that dsP21-322 arrested the cell cycle at the G0/G1 phase in HepG2 cells but at G2/M phase in Hep3B cells which lack functional p53 and Rb genes, and induced both early and late stage apoptosis by activating caspase 3 in both cell lines. These results demonstrated that RNAa of p21 has in vitro antigrowth effects on HCC cells via impeding cell cycle progression and inducing apoptotic cell death. This study suggests that targeted activation of p21 by RNAa may be explored as a novel therapy for the treatment of HCC.
PMCID: PMC3464408  PMID: 22909100
15.  Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells 
PLoS Genetics  2013;9(9):e1003821.
Argonaute proteins are often credited for their cytoplasmic activities in which they function as central mediators of the RNAi platform and microRNA (miRNA)-mediated processes. They also facilitate heterochromatin formation and establishment of repressive epigenetic marks in the nucleus of fission yeast and plants. However, the nuclear functions of Ago proteins in mammalian cells remain elusive. In the present study, we combine ChIP-seq (chromatin immunoprecipitation coupled with massively parallel sequencing) with biochemical assays to show that nuclear Ago1 directly interacts with RNA Polymerase II and is widely associated with chromosomal loci throughout the genome with preferential enrichment in promoters of transcriptionally active genes. Additional analyses show that nuclear Ago1 regulates the expression of Ago1-bound genes that are implicated in oncogenic pathways including cell cycle progression, growth, and survival. Our findings reveal the first landscape of human Ago1-chromosomal interactions, which may play a role in the oncogenic transcriptional program of cancer cells.
Author Summary
Argonaute (Ago) proteins are an evolutionarily conserved family of proteins indispensable for a gene regulation mechanism known as RNA interference (RNAi) which is mediated by small RNA including microRNA (miRNA) and small interfering RNA (siRNA) and occurs mainly in the cytoplasm. In mammalian cells, however, the function of Agos in the nucleus is largely unknown despite a few examples in which Agos are shown to be involved in regulating gene transcription and alternative splicing. In this study, by taking a genome-wide approach, we found that human Ago1, but not Ago2, is pervasively associated with gene regulatory sequences known as promoter and interacts with the core component of the gene transcription machinery to exert positive impact on gene expression in cancer cells. Strikingly, the genes bound and regulated by Ago1 are mostly genes that stimulate cell growth and survival, and are known to be involved in the development of cancer. The findings from our study unveil an unexpected role of nuclear Ago1 in regulating gene expression which may be important both in normal cellular processes and in disease such as cancer.
PMCID: PMC3784563  PMID: 24086155
16.  Innate immunity activation involved in unprotected porcine auto-transplant kidneys preserved by naked caspase-3 siRNA 
The naked caspase-3 small interfering RNA (siRNA) infused into the renal artery during cold preservation was effective, but did not protect auto-transplant porcine kidneys with increased inflammation and apoptosis in our previous study. The mechanisms involved, in particular, whether siRNA or complementary systemic feedback eliciting innate immune responses are worthy to be further investigated.
The protein and mRNA expression of innate immunity-related molecules were detected by western blotting and quantitative PCR in the tissues previously collected from 48 h auto-transplant kidneys. The donor kidneys were retrieved from mini pigs and cold preserved by University of Wisconsin solution with/without 0.3 mg caspase-3 siRNA for 24 h.
The protein level of Toll like receptor (TLR) 3, TLR7, and their main adapters, TRIF and MyD88, was up-regulated in the siRNA preserved auto-transplant kidneys. The mRNA level of NF-κB and c-Jun was increased, as well as pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α and interferon (IFN)-α, β and γ. In addition, the non-TLR RNA sensor PKR protein, but not RIG1, was also increased in the siRNA preserved auto-transplant kidneys.
The activation of innate immunity with amplified inflammatory responses in the caspase-3 siRNA preserved auto-transplant kidneys are associated with increased TLR3, TLR7 and PKR, which might be due to complementary systemic feedback, although persistent actions initiated by short-acting caspase-3 siRNA cannot be completely ruled out. These results provided valuable evidence to guide future siRNA design and pre-clinic studies.
PMCID: PMC3847504  PMID: 24034868
Small interfering RNA; Innate immunity; Toll like receptor; PKR; Porcine kidney auto-transplantation; Ischemia reperfusion injury
17.  Involvement of MicroRNAs in Infection of Silkworm with Bombyx mori Cytoplasmic Polyhedrosis Virus (BmCPV) 
PLoS ONE  2013;8(7):e68209.
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the most important pathogens of silkworm. MicroRNAs (miRNAs) have been demonstrated to play key roles in regulating host-pathogen interaction. However, there are limited reports on the miRNAs expression profiles during insect pathogen challenges. In this study, four small RNA libraries from BmCPV-infected midgut of silkworm at 72 h post-inoculation and 96 h post-inoculation and their corresponding control midguts were constructed and deep sequenced. A total of 316 known miRNAs (including miRNA*) and 90 novel miRNAs were identified. Fifty-eight miRNAs displayed significant differential expression between the infected and normal midgut (P value < = 0.01 and fold change > = 2.0 or < = 0.5), among which ten differentially expressed miRNA were validated by qRT-PCR method. Further bioinformatics analysis of predicted target genes of differentially expressed miRNAs showed that the miRNA targets were involved in stimulus and immune system process in silkworm.
PMCID: PMC3699532  PMID: 23844171
18.  Role of Cystathionine γ-Lyase/Hydrogen Sulfide Pathway in Cardiovascular Disease: A Novel Therapeutic Strategy? 
Antioxidants & Redox Signaling  2012;17(1):106-118.
Significance: Hydrogen sulfide (H2S) has traditionally been considered a toxic environmental pollutant. In the late 1990s, the presumed solely harmful role of H2S has been challenged because H2S may also be involved in the maintenance and preservation of cardiovascular homeostasis. Recent Advances: The production of endogenous H2S has been attributed to three key enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase. The recognition of H2S as the third gaseous signaling molecule has stimulated research on a multitude of pathophysiologic events in the cardiovascular system. In particular, important roles in cardiovascular disorder processes are ascribed to the CSE/H2S pathway, such as atherosclerosis, myocardial infarction, hypertension, and shock. Critical Issues: Many biological activities and molecular mechanisms of H2S in the cardiovascular system have been demonstrated in studies using different tools, such as the genetic overexpression of CSE, the direct administration of H2S donors, or the use of H2S-releasing pro-drugs. Unfortunately, the role of the CSE/H2S pathway in cardiovascular disease remains controversial in numerous areas, and many questions regarding the gaseous molecule still remain unanswered. Future Directions: Advances in basic research indicate that the CSE/H2S pathway may provide potential therapeutic targets for treating cardiovascular disorders. But the molecular targets of H2S still need to be identified. Antioxid. Redox Signal. 17, 106–118.
PMCID: PMC3342562  PMID: 22017202
19.  Loss of RASSF1A Expression in Colorectal Cancer and Its Association with K-ras Status 
BioMed Research International  2013;2013:976765.
Background. The RAS-association domain family 1 A (RASSF1A) is a classical member of RAS effectors regulating cell proliferation and apoptosis. Loss of RASSF1A expression may shift the balance towards a growth-promoting effect without the necessity of activating K-ras mutations. Its potential association with K-ras mutations in colorectal cancer (CRC) is unclear. Methods. RASSF1A expression was examined in normal mucosa, adenoma, and tumor tissues of colon and rectum, respectively. We examined the association of RASSF1A expression, mutations of K-ras, and EGFR status in 76 primary CRCs. The relationship between clinicopathological characteristics and RASSF1A expression was also analyzed. Results. RASSF1A expression level decreased progressively in normal mucosa, adenoma and, tumor tissues, and the loss of RASSF1A expression occurred more frequently in tumor tissues. Of 76 primary CRCs, loss of RASSF1A expression and/or K-ras mutations were detected in 77% cases. Loss of RASSF1A expression was more frequent in K-ras wild-type than in mutation cases (63% versus 32%, P = 0.011). Conclusions. Our study indicates that loss of RASSF1A may be involved in pathogenesis of CRC, its expression was found predominantly in K-ras wild-type CRCs, suggesting that it may be another way of affecting RAS signaling, in addition to K-ras mutations.
PMCID: PMC3705944  PMID: 23865079
20.  Splicing of Goose Parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA 
Virology  2012;426(1):60-65.
Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.
PMCID: PMC3294254  PMID: 22326101
21.  Epidermal Growth Factor Receptor (EGFR)-RAS Signaling Pathway in Penile Squamous Cell Carcinoma 
PLoS ONE  2013;8(4):e62175.
Penile Squamous Cell Carcinoma (SCC) is a rare cancer with poor prognosis and limited response to conventional chemotherapy. The genetic and epigenetic alterations of Epidermal Growth Factor Receptor (EGFR)-RAS-RAF signaling in penile SCC are unclear. This study aims to investigate four key members of this pathway in penile SCC. We examined the expression of EGFR and RAS-association domain family 1 A (RASSF1A) as well as the mutation status of K-RAS and BRAF in 150 cases of penile SCC. EGFR and RASSF1A expression was evaluated by immunohistochemistry. KRAS mutations at codons 12 and 13, and the BRAF mutation at codon 600 were analyzed on DNA isolated from formalin fixed paraffin embedded tissues by direct genomic sequencing. EGFR expression was positive in all specimens, and its over-expression rate was 92%. RASSF1A expression rate was only 3.42%. Significant correlation was not found between the expression of EGFR or RASSF1A and tumor grade, pT stage or lymph node metastases. The detection of KRAS and BRAF mutations analysis was performed in 94 and 83 tumor tissues, respectively. We found KRAS mutation in only one sample and found no BRAF V600E point mutation. In summary, we found over-expression of EGFR in the majority cases of penile SCC, but only rare expression of RASSF1A, rare KRAS mutation, and no BRAF mutation in penile SCC. These data suggest that anti-EGFR agents may be potentially considered as therapeutic options in penile SCC.
PMCID: PMC3634795  PMID: 23637996
22.  Differentiation of Central Lung Cancer from Atelectasis: Comparison of Diffusion-Weighted MRI with PET/CT 
PLoS ONE  2013;8(4):e60279.
Prospectively assess the performance of diffusion-weighted magnetic resonance imaging (DW-MRI) for differentiation of central lung cancer from atelectasis.
Materials and Methods
38 consecutive lung cancer patients (26 males, 12 females; age range: 28–71 years; mean age: 49 years) who were referred for thoracic MR imaging examinations were enrolled. MR examinations were performed using a 1.5-T clinical scanner and scanning sequences of T1WI, T2WI, and DWI. Cancers and atelectasis were measured by mapping of the apparent diffusion coefficients (ADCs) obtained with a b-value of 500 s/mm2.
PET/CT and DW-MR allowed differentiation of tumor and atelectasis in all 38 cases, but T2WI did not allow differentiation in 9 cases. Comparison of conventional T2WI and DW-MRI indicated a higher contrast noise ratio of the central lung carcinoma than the atelectasis by DW-MRI. ADC maps indicated significantly lower mean ADC in the central lung carcinoma than in the atelectasis (1.83±0.58 vs. 2.90±0.26 mm2/s, p<0.0001). ADC values of small cell lung carcinoma were significantly greater than those from squamous cell carcinoma and adenocarcinoma (p<0.0001 for both).
DW-MR imaging provides valuable information not obtained by conventional MR and may be useful for differentiation of central lung carcinoma from atelectasis. Future developments may allow DW-MR imaging to be used as an alternative to PET-CT in imaging of patients with lung cancer.
PMCID: PMC3617228  PMID: 23593186
23.  Cotton GalT1 Encoding a Putative Glycosyltransferase Is Involved in Regulation of Cell Wall Pectin Biosynthesis during Plant Development 
PLoS ONE  2013;8(3):e59115.
Arabinogalactan proteins (AGPs), are a group of highly glycosylated proteins that are found throughout the plant kingdom. To date, glycosyltransferases that glycosylate AGP backbone have remained largely unknown. In this study, a gene (GhGalT1) encoding a putative β-1,3-galactosyltransferase (GalT) was identified in cotton. GhGalT1, belonging to CAZy GT31 family, is the type II membrane protein that contains an N-terminal transmembrane domain and a C-terminal galactosyltransferase functional domain. A subcellular localization assay demonstrated that GhGalT1 was localized in the Golgi apparatus. RT-PCR analysis revealed that GhGalT1 was expressed at relatively high levels in hypocotyls, roots, fibers and ovules. Overexpression of GhGalT1 in Arabidopsis promoted plant growth and metabolism. The transgenic seedlings had much longer primary roots, higher chlorophyll content, higher photosynthetic efficiency, the increased biomass, and the enhanced tolerance to exogenous D-arabinose and D-galactose. In addition, gas chromatography (GC) analysis of monosaccharide composition of cell wall fractions showed that pectin was changed in the transgenic plants, compared with that of wild type. Three genes (GAUT8, GAUT9 and xgd1) involved in pectin biosynthesis were dramatically up-regulated in the transgenic lines. These data suggested that GhGalT1 may be involved in regulation of pectin biosynthesis required for plant development.
PMCID: PMC3601089  PMID: 23527103
24.  IKKα Activation of NOTCH LinksTumorigenesis via FOXA2 Suppression 
Molecular Cell  2011;45(2):171-184.
Pro-inflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/ S111, thereby suppressing FOXA2 transactivation activity, leading to decreased NUMB expression and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.
PMCID: PMC3268914  PMID: 22196886
25.  Insertion/Deletion Polymorphisms in the ΔNp63 Promoter Are a Risk Factor for Bladder Exstrophy Epispadias Complex 
PLoS Genetics  2012;8(12):e1003070.
Bladder exstrophy epispadias complex (BEEC) is a severe congenital anomaly; however, the genetic and molecular mechanisms underlying the formation of BEEC remain unclear. TP63, a member of TP53 tumor suppressor gene family, is expressed in bladder urothelium and skin over the external genitalia during mammalian development. It plays a role in bladder development. We have previously shown that p63−/− mouse embryos developed a bladder exstrophy phenotype identical to human BEEC. We hypothesised that TP63 is involved in human BEEC pathogenesis. RNA was extracted from BEEC foreskin specimens and, as in mice, ΔNp63 was the predominant p63 isoform. ΔNp63 expression in the foreskin and bladder epithelium of BEEC patients was reduced. DNA was sequenced from 163 BEEC patients and 285 ethnicity-matched controls. No exon mutations were detected. Sequencing of the ΔNp63 promoter showed 7 single nucleotide polymorphisms and 4 insertion/deletion (indel) polymorphisms. Indel polymorphisms were associated with an increased risk of BEEC. Significantly the sites of indel polymorphisms differed between Caucasian and non-Caucasian populations. A 12-base-pair deletion was associated with an increased risk with only Caucasian patients (p = 0.0052 Odds Ratio (OR) = 18.33), whereas a 4-base-pair insertion was only associated with non-Caucasian patients (p = 0.0259 OR = 4.583). We found a consistent and statistically significant reduction in transcriptional efficiencies of the promoter sequences containing indel polymorphisms in luciferase assays. These findings suggest that indel polymorphisms of the ΔNp63 promoter lead to a reduction in p63 expression, which could lead to BEEC.
Author Summary
Bladder exstrophy epispadias complex is a severe congenital abnormality. The affected babies' bladders are born open, leaking urine constantly. Treatment involves multiple major reconstructive surgeries and the need for lifelong care for the complications of the disease. Although a number of studies have suggested a genetic cause of the disease, the genetic and molecular mechanism underlying the formation of BEEC remains unknown. One gene, TP63, plays a crucial role in the early bladder development. Two different genetic promoters of TP63 produce different forms of the protein with opposing properties. We have shown mice lacking p63 displayed a deformity complex identical to human BEEC. There are no genetic mutations in the p63 protein in BEEC, so genetic variants in the promoter could alter protein expression. Our hypothesis was that loss of p63 expression due to sequence polymorphisms in a promoter is a risk factor for BEEC. We found promoter sequence variants that were statistically associated with the disease and the sequence variant location varied between Caucasian and non-Caucasian patients. This is particularly important as Caucasian populations have a higher risk of BEEC. These findings provide an explanation of BECC and a base for further study of TP63 related genes in this disease.
PMCID: PMC3527294  PMID: 23284286

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