Differences in silencing of mismatched targets by sliced versus diced siRNAs.

It has been reported that the two major types of RNA interference triggers, the classical Dicer-generated small RNAs (siRNAs), which function with all members of the Argonaute (Ago) protein family in mammals, and the Ago2-sliced small RNAs (sli-siRNAs), which function solely through Ago2, have similar potency in target cleavage and repression. Here, we show that sli-siRNAs are generally more potent than siRNAs in silencing mismatched targets. This phenomenon is usually more apparent in targets that have mismatched nucleotides in the 3' supplementary region than in targets with mismatches in the seed region. We demonstrate that Ago2 slicer activity is a major factor contributing to the greater silencing efficiency of sli-siRNA against mismatched targets and that participation of non-slicing Agos in silencing mismatched siRNA targets may dilute the slicing ability of Ago2. The difference in length of the mature guide RNA used in sli-RISCs and si-RISCs may also contribute to the observed difference in knockdown efficiency. Our data suggest that a sli-siRNA guide strand is likely to have substantially stronger off-target effects than a guide strand with the same sequence in a classical siRNA and that Dicer and non-slicing Agos may play pivotal roles in controlling siRNA target specificity.

Targeting miR-193a-AML1-ETO-ß-catenin axis by melatonin suppresses the self-renewal of leukaemia stem cells in leukaemia with t (8;21) translocation.

AML1-ETO, the most common fusion oncoprotein by t (8;21) in acute myeloid leukaemia (AML), enhances hematopoietic self-renewal and leukemogenesis. However, currently no specific therapies have been reported for t (8;21) AML patients as AML1-ETO is still intractable as a pharmacological target. For this purpose, leukaemia cells and AML1-ETO-induced murine leukaemia model were used to investigate the degradation of AML1-ETO by melatonin (MLT), synthesized and secreted by the pineal gland. MLT remarkedly decreased AML1-ETO protein in leukemic cells. Meanwhile, MLT induced apoptosis, decreased proliferation and reduced colony formation. Furthermore, MLT reduced the expansion of human leukemic cells and extended the overall survival in U937T-AML1-ETO-xenografted NSG mice. Most importantly, MLT reduced the infiltration of leukaemia blasts, decreased the frequency of leukaemia stem cells (LSCs) and prolonged the overall survival in AML1-ETO-induced murine leukaemia. Mechanistically, MLT increased the expression of miR-193a, which inhibited AML1-ETO expression via targeting its putative binding sites. Furthermore, MLT decreased the expression of ß-catenin, which is required for the self-renewal of LSC and is the downstream of AML1-ETO. Thus, MLT presents anti-self-renewal of LSC through miR-193a-AML1-ETO-ß-catenin axis. In conclusion, MLT might be a potential treatment for t (8;21) leukaemia by targeting AML1-ETO oncoprotein.

Dovitinib synergizes with oxaliplatin in suppressing cell proliferation and inducing apoptosis in colorectal cancer cells regardless of RAS-RAF mutation status.

BACKGROUND: Cancer is the result of a multistep process of genomic alterations, including mutations in key regulatory proteins that result in loss of balanced gene expression and subsequent malignant transformation. Throughout the various stages of colorectal carcinoma (CRC), complex genetic alterations occur, of which over-expression of growth factors, such as vascular endothelial growth factor, fibroblast growth factor and platelet-derive growth factor and their corresponding receptor tyrosine kinases, have been shown to correlate with invasiveness, tumor angiogenesis, metastasis, recurrence, and poor prognosis of colorectal cancer. To evaluate the therapeutic effect, we combined Dovitinib, an orally bioavailable, potent inhibitor of class III-V receptor tyrosine kinases with chemotherapeutic drug, oxaliplatin in preclinical models of colon cancer. METHODS: Human colon cancer cells with different RAS-RAF mutation status (HCT-116, HT-29, SW-480, CaCO2 and LS174T) were treated with a combination of Dovitinib and Oxaliplatin at low dosage followed by assays to investigate the effect of the combination on cell proliferation, cell migration, cell apoptosis and signaling pathways involved in molecular mechanism of drug(s). The antitumor effects of either of the drugs were compared to the combination using human colon carcinoma cell line HT-29 xenograft model. Treated vs untreated tumor sections were also compared for proliferation and angiogenesis markers by immunohistochemistry. RESULTS: The combination of dovitinib and oxaliplatin showed higher in vitro cytotoxicity in colon cell lines irrespective of their RAS-RAF status as compared to either of the drugs alone. Simultaneous inhibition of MAP kinase and AKT pathways and induction of apoptosis via activation of caspases 9/caspases 3 contributed to the synergistic effect of this combination therapy. In the xenograft model, the combination showed a significantly higher antitumor activity. Immunohistochemistry of post treatment tumors showed a significant decrease in proliferation and angiogenesis as compared to either of the treatments alone. CONCLUSIONS: This study demonstrates the synergistic antitumor activity of combination of dovitinib and oxaliplatin against colon cancer with different RAS-RAF status. The combination also showed its antitumor efficacy in a multidrug resistant phenotype xenograft model. This provides a basis for further investigation for its potential in clinical setting for colorectal cancer.

Dehydroascorbic acid taken up by glucose transporters stimulates estradiol production through inhibition of JNK/c-Jun/AP1 signaling in JAR cells.

We have previously demonstrated that the reduced form of vitamin C (l-ascorbic acid, AA) is able to induce the production of both steroid and peptide hormones in human choriocarcinoma cells. Here, we attempted to investigate the role and underlying mechanism of the oxidized form of vitamin C, dehydroascorbic acid (DHA), in steroidogenesis in primary human cytotrophoblasts and human choriocarcinoma cells. Messenger RNA and protein levels of steroidogenic enzymes including P450 cholesterol side-chain cleavage enzyme (P450scc), 3ß-hydroxysteroid dehydrogenase type 1 (3ß-HSD1), 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) and aromatase were examined by quantitative RT-PCR and western blots, respectively. Progesterone (P4) and estradiol (E2) levels were determined by enzyme immunoassays. Knockdown of c-Jun was achieved by lentivirus-mediated shRNA, and signaling pathways implicated in DHA-induced steroidogenesis were examined by western blots and dual-luciferase assays. DHA dose-dependently induced the expression of steroidogenic enzymes including 3ß-HSD1, 17ß-HSD1 and aromatase at both mRNA and protein levels, and subsequently increased the production of E2 but not P4. These effects were synergized by diethylmaleate, a glutathione-depleting compound, and α-tocopherol, a reducing agent, but robustly attenuated by inhibition of DHA transportation by phloretin or 2-deoxy-d-glucose. DHA time-dependently inhibited JNK and c-Jun phosphorylation, and dose-dependently reduced AP1 reporter activity. JNK signaling pathway-specific inhibitor SP600125 and c-Jun shRNA both significantly increased the expression of steroidogenic enzymes and E2 production regardless of the presence or absence of DHA. These findings suggest that DHA is able to induce steroidogenesis through inhibition of JNK/c-Jun/AP1 signaling, and may therefore play indispensable roles in pregnancy maintenance.

MicroRNA-657 promotes tumorigenesis in hepatocellular carcinoma by targeting transducin-like enhancer protein 1 through nuclear factor kappa B pathways.

UNLABELLED: Growing evidence indicates that deregulation of microRNAs (miRNAs) contributes to tumorigenesis. Dysregulation of miR-657 has been observed in several types of cancers, but its biological function is still largely unknown. Our results showed that miR-657 expression can be induced by hepatitis viral proteins and is significantly increased in hepatocellular carcinoma (HCC) tissues. Moreover, introduction of miR-657 dramatically increases proliferation and colony formation of HCC cells in vitro and induces tumor development in immunodeficient mice. Further studies showed that miR-657 directly targets the transducin-like enhancer protein 1 (TLE1) 3' untranslated region (UTR) and activates nuclear factor kappa B (NF-κB) pathways that contribute to hepatocarcinogenesis. CONCLUSION: This study identified a mechanism whereby miRNA-657 contributed to HCC through novel cancer pathways and provides new insights into the potential molecular mechanisms of hepatic carcinogenesis.

Pharmacodynamic and pharmacogenomic study of the nanoparticle conjugate of camptothecin CRLX101 for the treatment of cancer.

CRLX101 is a nanopharmaceutical consisting of cyclodextrin-based polymer molecule and camptothecin. The CRLX101 nanoparticle is designed to concentrate and slowly release camptothecin in tumors over an extended period of time. Tumor biopsy and blood samples collected from patients with advanced solid malignancies before and after CRLX101 treatment are subjected to immunohistochemistry and pharmacogenomics. The expression of Topoisomerase-1, Ki-67, CaIX, CD31 and VEGF decreased after CRLX101 treatment. The expressions of these proteins are inversely proportional with survival duration of the patients. The Drug Metabolism Enzymes and Transporters (DMET) array shows an allele frequency in patients similar to global populations with none of the SNPs associated with toxicity. The results suggest that the observed lower toxicity is not likely to be due to different genotypes in SNPs. CRLX101 demonstrates a promising anti-tumor activity in heavily pre-treated or treatment-refractory solid tumor malignancies presumably by inhibition of proliferation and angiogenesis correlating with tumor growth inhibition. From the clinical editor: In this cancer treatment study clinical samples collected from patients were subjected to immunohistochemistry and pharmacogenomics. The expressions of key proteins that are inversely proportional with survival duration of the patients decreased after treatment with CRLX101, a camptothecin slow-release nanoparticle conjugate. This anti-tumor activity in heavily pre-treated and treatment resistant solid tumors, promises a novel therapeutic approach.

Understanding the mechanism of acupuncture in acute cerebral infraction through a proteomic analysis: protocol for a prospective randomized controlled trial.

Background: Acute cerebral infarction (ACI), being the predominant form of stroke, presents challenges in terms of the limited effectiveness of various treatments in improving the neurological function. Although acupuncture shows promise in addressing ACI, the availability of high-quality evidence regarding its efficacy, safety, and underlying mechanism remains insufficient. In this study, we design a multicenter, prospective, single-blind, randomized controlled trial with the aim of evaluating the efficacy and safety of acupuncture for ACI, making an attempt to unveil the molecular mechanisms by proteomic. Methods: A total of 132 patients involving four hospitals will be randomized at a 1:1:1 ratio in the acupuncture group, control group, and sham acupuncture group. All the patients will receive basic treatment, and the patients in the acupuncture and sham acupuncture groups will also receive either acupuncture or sham acupuncture treatment, respectively, at six sessions each week for a 2 weeks period, followed by 3 months of follow-up. The primary outcome will be the change in the National Institute of Health Stroke Scale (NIHSS) scores after treatment. The secondary outcomes will include the Fugl-Meyer Assessment (FMA) scale scores and the Barthel Index (BI). Adverse events that occur during the trial will be documented. To discover differentially expressed proteins (DEPs) and their roles between the ACI subjects and healthy controls, we will also perform 4D-DIA quantitative proteomics analysis, and the DEPs will be confirmed by enzyme-linked immunosorbent assay (ELISA). This study was approved by the institutional review board of the First Teaching Hospital of Tianjin University of Traditional Chinese Medicine (TYLL2023043). Written informed consent from patients is required. This trial is registered in the Chinese Clinical Trial Registry (ChiCTR2300079204). Trial results will be published in a peer-reviewed academic journal. Discussion: The results of this study will determine the preliminary efficacy and safety of acupuncture in ACI patients and whether the mechanism of this form of non-pharmacologic stimulation is mediated by a novel therapeutic target for neurorehabilitation through our proteomic analysis. Clinical trial registration: https://www.chictr.org.cn, identifier ChiCTR2300079204.

Understanding the Relationship Between Vascular Smooth Muscle Cell Function and the Efficacy of Acupuncture in Treating Cerebral Ischemic Stroke: A Preclinical Meta-Analysis and Systematic Review.

Background: Cerebral blood flow and vascular structures serve as the fundamental components of brain metabolism and circulation. Acupuncture, an alternative and complementary medical approach, has demonstrated efficacy in treating cerebral ischemic stroke (CIS). Nevertheless, the mechanisms underlying the impact of acupuncture on vascular smooth muscle cell (VSMC) function remain uncertain. The objective of this systematic review and meta-analysis is to assess the alterations in VSMC function following acupuncture stimulation in CIS models. Methods: The databases PubMed, Web of Science, SCOPUS, and EMBASE were queried until November 2022 using a predetermined search strategy. The FORMAT BY SYRCLE guidelines were adhered to, and the risk of bias of the included studies was evaluated using the Risk of Bias tool developed by the Systematic Review Centre for Laboratory Animal Experimentation. The random-effects model was employed to estimate the standardized mean difference (SMD). Results: Eighteen articles are included in this review. Acupuncture showed significant positive effects on the region cerebral blood flow (SMD=8.15 [95% CI, 4.52 to 11.78]) and neurological deficiency (SMD=-3.75 [95% CI, -5.54 to -1.97]). Descriptive analysis showed a probable mechanism of acupuncture stimulation in CIS rats related to VSMC function. Limitations and publication bias were presented in the studies. Conclusion: In this systematic review and meta-analysis, our findings indicate that acupuncture stimulation has the potential to improve regional cerebral blood flow and alleviate neurological deficits, possibly by regulating VSMC function. However, it is important to exercise caution when interpreting these results due to the limitations of animal experimental design and methodological quality.

Extracellular vesicles and their indispensable roles in pathogenesis and treatment of inflammatory bowel disease: A comprehensive review.

Inflammatory bowel disease (IBD) is a global disease with rising incidence worldwide, and its debilitating symptoms and dissatisfactory therapies have brought heavy burdens for patients. Extracellular vesicles (EVs), a heterogeneous population of lipid bilayer membranes containing abundant bioactive molecules, have been indicated to play important roles in the pathogenesis and treatment of many diseases. However, to our knowledge, comprehensive reviews summarizing the various roles of diverse source-derived EVs in the pathogenesis and treatment of IBD are still lacking. This review, not only summarizes the EV characteristics, but also focuses on the multiple roles of diverse EVs in IBD pathogenesis and their treatment potential. In addition, hoping to push forward the research frontiers, we point out several challenges that the researchers are faced, about EVs in current IBD research and future therapeutic applications. We also put forward our prospects on future exploration regarding EVs in IBD treatment, including developing IBD vaccines and paying more attention on apoptotic vesicles. This review is aimed to enrich the knowledge on the indispensable roles of EVs in IBD pathogenesis and treatment, providing ideas and reference for future therapeutic strategy for IBD treatment.

Preclinical study of the cyclodextrin-polymer conjugate of camptothecin CRLX101 for the treatment of gastric cancer.

Camptothecin showed remarkable anticancer activity in animal models of cancer but was restricted in clinical use for its adverse toxicity in patients. The preclinical efficacy of CRLX101, a nanoparticle (NP) assembly containing cyclodextrin-based polymer and camptothecin was evaluated by in vitro cytotoxicity in gastric cancer cell lines and in vivo antitumor effects in human gastric cancer cell line BGC823 xenografts. Treated tumor sections were analyzed for presence of NPs and compared with vehicle control tumors for hypoxia and angiogenesis. Gastric cancer cell lines showed high in vitro cytotoxicity for CRLX101 and also showed strong antitumor activity in vivo. Electron micrographs revealed the intracellular presence of NPs in close proximity to vesicles. A significant decrease in expressions of carbonic anhydrase, VEGF, and CD31 proteins in treated tumors indicated an inhibition of hypoxia and angiogenesis. The results provide preclinical data for gastric adenocarcinoma. FROM THE CLINICAL EDITOR: This study describes a nanoparticle assembly containing cyclodextrin-based polymer and camptothecin, resulting in increased bioavailability of camptothecin, an effective but toxic anti-cancer agent. The antitumor effects and safety profile were demonstrated in a gastric carcinoma cell line.

The effects of maxillary protraction and its long-term stability--a clinical trial in Chinese adolescents.

The purpose of this study was to evaluate the effect of reverse pull headgear (RPHG) in the treatment of Class III malocclusions in the late mixed and early permanent dentition and its long-term stability at the time when facial growth was close to completion. The treatment group comprised 22 subjects (12 males and 10 females, mean age: 11.38 ± 0.69 years). The control group included 17 subjects (7 males and 10 females, mean age: 11.54 ± 1.07 years). The mean RPHG treatment time was 1.5 ± 0.95 years and the observation time for the control group was 1.75 ± 0.83 years. For each subject, lateral cephalograms were obtained before (T1) and after (T2) treatment or observation. These cephalograms were traced and analyzed and the differences between T1 and T2 values were examined with paired t-tests. Of the 22 treated cases, 10 patients were followed up until at the mean age of 16.18 years (T3). Since there was no relapse in anterior crossbite, the long-term effects of RPHG were evaluated by measuring the maxillary and mandibular skeletal changes. The follow-up patients were divided into two groups based on the change in ANB: a stable group (decrease in ANB < 2 degrees) and an unstable group (decrease in ANB > 2 degrees). The skeletal effects of RPHG in treating Class III anomalies just before or at the beginning of the pubertal growth spurt include protraction of the maxilla and dentition and inhibition of forward growth of the mandible. With regard to the long-term change, a slight alteration in the position of the maxilla and in the position and growth direction of the mandible resulted in a slight decrease in ANB in the stable group. The slight retrusion in the maxilla, combined with the significant protrusion in the mandible and the more horizontal mandibular growth direction, resulted in a decrease in ANB in the unstable group. This indicated that the maxilla remained relatively stable and that the unstable factor was continuing mandibular growth during the pubertal and post-pubertal period. For patients with an excessive mandible, orthopaedic therapy should start at the beginning of pubertal growth and orthodontic fixed appliance should follow immediately after RPHG so that mandibular growth in the sagittal direction during puberty or even after pubertal growth may be effectively inhibited.

The Effects of Alda-1 Treatment on Renal and Intestinal Injuries After Cardiopulmonary Resuscitation in Pigs.

Aim: After successful cardiopulmonary resuscitation (CPR), most survivors will develop acute kidney injury and intestinal barrier dysfunction, both of which contribute to the poor outcomes of cardiac arrest (CA) victims. Recently, the aldehyde dehydrogenase 2 (ALDH2) agonist, Alda-1 was shown to effectively alleviate regional ischemia/reperfusion injury of various organs. In the present study, we investigated the effects of Alda-1 treatment on renal and intestinal injuries after CA and resuscitation in pigs. Methods: Twenty-four male domestic pigs were randomly divided into one of the three groups: sham (n = 6), CPR (n = 10), or CPR+Alda-1 (n = 8). CA was induced and untreated for 8 min, and then CPR was performed for 8 min in the CPR and CPR+Alda-1 groups. At 5 min after resuscitation, a dose of 0.88 mg/kg of Alda-1 was intravenously administered in the CPR+Alda-1 group. The biomarkers of renal and intestinal injuries after resuscitation were regularly measured for a total of 24 h. Subsequently, the animals were euthanized, and then renal and intestinal tissues were obtained for the measurements of ALDH2 activity and expression, and cell apoptosis and ferroptosis. Results: Five of the 10 animals in the CPR group and six of the eight animals in the CPR+Alda-1 group were successfully resuscitated. After resuscitation, the levels of biomarkers of renal and intestinal injuries were significantly increased in all animals experiencing CA and resuscitation compared with the sham group; however, Alda-1 treatment significantly alleviated renal and intestinal injuries compared to the CPR group. Post-resuscitation ALDH2 activity was significantly decreased and its expression was markedly reduced in the kidney and intestine in those resuscitated animals compared with the sham group; nevertheless, both of them were significantly greater in those animals receiving Alda-1 treatment compared to the CPR group. In addition, renal, intestinal apoptosis and ferroptosis after resuscitation were observed in the CPR and CPR+Alda-1 groups, in which both of them were significantly milder in the CPR+Alda1 group than in the CPR group. Conclusions: The activation of ALDH2 by Alda-1 treatment significantly alleviated post-resuscitation renal and intestinal injuries through the inhibition of cell apoptosis and ferroptosis in a pig model of CA and resuscitation.

[Effect of manual acupuncture of "Shuigou" (GV26)"Neiguan" (PC6) on neurological function and expression of apoptosis-related factors in brain tissues surrounding hematoma in intracerebral hemorrhage rats].

OBJECTIVE: To observe the effect of manual acupuncture stimulation of "Shuigou" (GV26) and "Neiguan" (PC6) on neurological function and expression of Caspase-3 and Caspase-9 in brain tissues around the intracerebral hematoma in rats with acute intracerebral hemorrhage (ICH), so as to explore its possible mechanisms underlying improvement of ICH. METHODS: Ninety-six male SD rats were randomly divided into 4 groups: control, model, acupoint and non-acupoint (24 rats in each group). The ICH model was established by injection of the rat's autologous blood into the caudate nucleus. According to the time-points of 6, 24, 48 and 72 h after ICH, each of the 4 groups was further divided into 4 subgroups. For rats of the acupoint group, the PC6 on both sides was manually stimulated by manipulating the needle with lifting-thrusting-twisting reducing techniques, while the GV26 was stimulated with strong "sparrow-pecking" method for 10 times, then, left the needles in the acupoints for 30 min. For rats of the non-acupoint group, two non-acupoints: mid-spot below the bilateral axilla and the spot 3 mm above the left side of the coccyx tip were stimulated with the same methods to PC6 and GV26, respectively. For rats of the 6 h and 24 h subgroups, the intervention was given once after waking up from modeling, and for those of the 48 and 72 h subgroups, the intervention was conducted once a day for 2 or 3 times, respectively. The neurological severity score (NSS) was used to evaluate the degree of neurological function. The immunoactivity (expression) of Caspase-3 and Caspase-9 proteins of the hematoma focus of the brain was detected by immunohistochemistry. RESULTS: Following modeling, the NSS and the expression levels of Caspase-3 and Caspase-9 proteins in the brain tissues surrounding the hematoma at each time-points (6, 24, 48 and 72h) after modeling were significantly increased in the model group relevant to the control group (P<0.01, P<0.05). Compared with the model group, the NSS at 72h and the expression levels of Caspase-3 and Caspase-9 proteins at 6, 24, 48 and 72h were significantly down-regulated in the acupoint group (P<0.05) rather than in the non-acupoint group (P>0.05). CONCLUSION: Acupuncture of GV26 and PC6 can improve the neurological function in rats with ICH, which may be related to its function in reducing the expression of Caspase-3 and Caspase-9 proteins (apoptosis-related proteins) in the brain.

Effects of the tropospheric large-scale circulation on European winter temperatures during the period of amplified Arctic warming.

We investigate factors influencing European winter (DJFM) air temperatures for the period 1979-2015 with the focus on changes during the recent period of rapid Arctic warming (1998-2015). We employ meteorological reanalyses analysed with a combination of correlation analysis, two pattern clustering techniques, and back-trajectory airmass identification. In all five selected European regions, severe cold winter events lasting at least 4 days are significantly correlated with warm Arctic episodes. Relationships during opposite conditions of warm Europe/cold Arctic are also significant. Correlations have become consistently stronger since 1998. Large-scale pattern analysis reveals that cold spells are associated with the negative phase of the North Atlantic Oscillation (NAO-) and the positive phase of the Scandinavian (SCA+) pattern, which in turn are correlated with the divergence of dry-static energy transport. Warm European extremes are associated with opposite phases of these patterns and the convergence of latent heat transport. Airmass trajectory analysis is consistent with these findings, as airmasses associated with extreme cold events typically originate over continents, while warm events tend to occur with prevailing maritime airmasses. Despite Arctic-wide warming, significant cooling has occurred in northeastern Europe owing to a decrease in adiabatic subsidence heating in airmasses arriving from the southeast, along with increased occurrence of circulation patterns favouring low temperature advection. These dynamic effects dominated over the increased mean temperature of most circulation patterns. Lagged correlation analysis reveals that SCA- and NAO+ are typically preceded by cold Arctic anomalies during the previous 2-3 months, which may aid seasonal forecasting.

Overexpression of RRM2 decreases thrombspondin-1 and increases VEGF production in human cancer cells in vitro and in vivo: implication of RRM2 in angiogenesis.

BACKGROUND: In addition to its essential role in ribonucleotide reduction, ribonucleotide reductase (RNR) small subunit, RRM2, has been known to play a critical role in determining tumor malignancy. Overexpression of RRM2 significantly enhances the invasive and metastatic potential of tumor. Angiogenesis is critical to tumor malignancy; it plays an essential role in tumor growth and metastasis. It is important to investigate whether the angiogenic potential of tumor is affected by RRM2. RESULTS: We examined the expression of antiangiogenic thrombospondin-1 (TSP-1) and proangiogenic vascular endothelial growth factor (VEGF) in two RRM2-overexpressing KB cells: KB-M2-D and KB-HURs. We found that TSP-1 was significantly decreased in both KB-M2-D and KB-HURs cells compared to the parental KB and mock transfected KB-V. Simultaneously, RRM2-overexpressing KB cells showed increased production of VEGF mRNA and protein. In contrast, attenuating RRM2 expression via siRNA resulted in a significant increased TSP-1 expression in both KB and LNCaP cells; while the expression of VEGF by the two cells was significantly decreased under both normoxia and hypoxia. In comparison with KB-V, overexpression of RRM2 had no significant effect on proliferation in vitro, but it dramatically accelerated in vivo subcutaneous growth of KB-M2-D. KB-M2-D possessed more angiogenic potential than KB-V, as shown in vitro by its increased chemotaxis for endothelial cells and in vivo by the generation of more vascularized tumor xenografts. CONCLUSION: These findings suggest a positive role of RRM2 in tumor angiogenesis and growth through regulation of the expression of TSP-1 and VEGF.

Superb nucleotide discrimination by a novel on/off switch for DNA polymerization and its applications.

With the use of polymerases having 3' to 5' exonuclease activity and 3' phosphorothioate-modified allele-specific primers, we recently devised a SNP-operated on/off switch controlling DNA polymerization. One advantage of this novel on/off switch is its adaptability to arrayed primer extension. To further expand its application in genetic analysis, this new on/off switch was evaluated in discrimination of the match/mismatch status of single nucleotides upstream from the primer 3' terminal. A set of seven amplicons was developed with the templates differing from each other by a single nucleotide. Using this set of amplicons, the new on/off switch was shown to be able to efficiently discriminate single nucleotide polymorphisms from the primer 3' terminus to the -6 position from the primer 3' terminus. These data, illustrating the broad single nucleotide discrimination ability of this novel on/off switch, explain why the SNP-operated on/off switch is powerful in SNP analysis, and also indicate useful applications to genetic analysis additional to SNP assay. First, these data broaden the application of the novel on/off switch in the analysis of mutations other than SNPs. Second, it raises a nucleotide-walking algorithm suitable for de novo array-based sequencing analysis.

[Novel mechanism of 3' exonuclease of polymerase in maintenance of DNA replication fidelity and its application in SNP assay].

Polymerase with 3' to 5'exonulcease plays an important role in the maintenance of in vivo DNA replication fidelity. In order to develop more reliable SNP assays, we revisit the underlying molecular mechanisms by which DNA polymerases with 3' exonucleases maintain high fidelity of DNA replication. In addition to mismatch removal by proofreading, we recently discovered a premature termination of polymerization by a new mechanism of OFF-switch. This novel ON/OFF switch turns off DNA polymerization from mismatched primers and turns on DNA polymerization from matched primers. Two SNP assays were developed based on the proofreading and the newly identified OFF-switch respectively: terminal labeled primer extension and the ON/OFF switch operated SNP assay. These two new methods are well adapted to conventional techniques such as electrophoresis, real time PCR, microplates, and microarray. Application of these reliable SNP assays will greatly facilitate genetic and biomedical studies in the post-genome era.

De novo synthesis of PCR templates for the development of SARS diagnostic assay.

A novel coronavirus was identified as the cause for severe acute respiratory syndrome (SARS). The complete sequence of SARS genome has provided an opportunity for the development of molecular diagnostic assays. To restrain further outbreak of SARS, the World Health Organization has posted several pairs of polymerase chain reaction (PCR) primers for early diagnosis and urged more research to be done on PCR protocols. Here we report a strategy for the de novo synthesis of PCR templates complimentary to the SARS virus genome, which has the advantage of working on PCR templates without concern about viral infection and also has the advantage that it can be used by those who do not have access to the SARS virus. This highly efficient and safe strategy for obtaining SARS gene fragments is useful for the development of PCR assays, as well as for the preparation of reliable positive controls for PCR testing kits.

On/off switch mediated by Exo+ polymerases: experimental analysis for its physiological and technological implications.

The potential physiological role and technological application of the premature termination of DNA polymerization through the off-switch of exo+ polymerases were studied using 3' phosphorothioate-modified or unmodified primers with single base mismatch distal to the 3' terminus. With exonuclease-digestible unmodified primers, a gradient premature termination of DNA polymerization was observed when amplified with exo+ polymerases. With 3' allele specific phosphorothioate-modified primers, an efficient off-switch effect occurred in the discrimination of a single nucleotide polymorphism when directly using genomic DNA. Clearly, the off-switch of exo+ polymerases is useful in biomedical research.

[Effect of 3' exonuclease activity of polymerase on extension of phosphorothioate-modified primers].

OBJECTIVE: To determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase. METHODS: Two-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated. RESULTS: Exo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase. CONCLUSION: These data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.