Sea Buckthorn Polyphenols Alleviate High-Fat-Diet-Induced Metabolic Disorders in Mice via Reprograming Hepatic Lipid Homeostasis Owing to Directly Targeting Fatty Acid Synthase.

Our previous studies found that Sea Buckthorn polyphenols (SBP) extract inhibits fatty acid synthase (FAS) in vitro. Thus, we continued to explore possible effects and underlying mechanisms of SBP on complicated metabolic disorders in long-term high-fat-diet (HFD)-fed mice. To reveal that, an integrated approach was developed in this study. Targeted quantitative lipidomics with a total of 904 unique lipids mapping contributes to profiling the comprehensive features of disarranged hepatic lipid homeostasis and discovering a set of newfound lipid-based biomarkers to predict the occurrence and indicate the progression of metabolic disorders beyond current indicators. On the other hand, technologies of intermolecular interactions characterization, especially surface plasmon resonance (SPR) assay, contribute to recognizing targeted bioactive constituents present in SBP. Our findings highlight hepatic lipid homeostasis maintenance and constituent-FAS enzyme interactions, to provide new insights that SBP as a functional food alleviates HFD-induced metabolic disorders in mice via reprograming hepatic lipid homeostasis caused by targeting FAS, owing to four polyphenols directly interacting with FAS and cinaroside binding to FAS with good affinity.

Surprisingly Popular-Based Adaptive Memetic Algorithm for Energy-Efficient Distributed Flexible Job Shop Scheduling.

With the development of the economy, distributed manufacturing has gradually become the mainstream production mode. This work aims to solve the energy-efficient distributed flexible job shop scheduling problem (EDFJSP) while simultaneously minimizing makespan and energy consumption. Some gaps are stated following: 1) the previous works usually adopt the memetic algorithm (MA) with variable neighborhood search. However, the local search (LS) operators are inefficient due to strong randomness; 2) the confidence-based adaptive operator selection model follows the experiences of the major crowds, which ignores the efficient operators with low weight, so it can not select the really efficient operator; 3) the previous works lack of efficient strategy to save energy; and 4) the mainstream memetic framework adopts LS to all solutions, which causes the population to converge too quickly and the diversity is extremely reduced. Thus, we propose a surprisingly popular-based adaptive MA (SPAMA) to overcome the above deficiencies. The contributions are as follows: 1) four problem-based LS operators are employed to improve the convergence; 2) a surprisingly popular degree (SPD) feedback-based self-modifying operators selection model is proposed to find the efficient operators with low weight and correct crowd decision making; 3) the full active scheduling decoding is presented to reduce the energy consumption; and 4) an elite strategy is designed to balance the resources between global and LS. In order to evaluate the effectiveness of SPAMA, it is compared with state-of-the-art algorithms on Mk and DP benchmarks. The results demonstrate the superiority of SPAMA to the state-of-art algorithms for solving EDFJSP.

Discovery of novel indole and indoline derivatives against Candida albicans as potent antifungal agents.

With the widespread use of azole antifungals in the clinic, the drug resistance has been emerging continuously. In this work, we have designed and prepared a series of novel indole and indoline derivatives, and in vitro antifungal activity against C. albicans were evaluated. The results showed that title compounds exhibited good antifungal effect on Azole-resistant C. albicans. Further mechanism study demonstrated that S18 could inhibit the biofilm formation and hyphae growth of C. albicans through the Ras-cAMP-PKA signaling pathway.

Molecular dissection on inhibition of Ras-induced cellular senescence by small t antigen of SV40.

Simian virus 40 (SV40) is a potentially oncogenic virus of monkey origin. Transmission, prevalence, and pathogenicity rates of SV40 are unclear, but infection can occur in humans, for example individuals with high contact with rhesus macaques and individuals that received contaminated early batches of polio vaccines in 1950-1963. In addition, several human polyomaviruses, proven carcinogenic, are also highly common in global populations. Cellular senescence is a major mechanism of cancer prevention in vivo. Hyperactivation of Ras usually induces cellular senescence rather than cell transformation. Previous studies suggest small t antigen (ST) of SV40 may interfere with cellular senescence induced by Ras. In the current study, ST was demonstrated to inhibit Ras-induced cellular senescence (RIS) and accumulation of DNA damage in Ras-activated cells. In addition, ST suppressed the signal transmission from BRaf to MEK and thus blocked the downstream transmission of the activated Ras signal. B56γ knockdown mimicked the inhibitory effects of ST overexpression on RIS. Furthermore, KSR1 knockdown inhibited Ras activation and the subsequent cellular senescence. Further mechanism studies indicated that the phosphorylation level of KSR1 rather than the levels of the total protein regulates the activation of Ras signaling pathway. In sum, ST inhibits the continuous hyperactivation of Ras signals by interfering with the normal functions of PP2A-B56γ of dephosphorylating KSR1, thus inhibiting the occurrence of cellular senescence. Although the roles of SV40 in human carcinogenesis are controversial so far, our study has shown that ST of polyomaviruses has tumorigenic potential by inhibiting oncogene-induced senescence (OIS) as a proof of concept.

System dynamics-based prediction of municipal solid waste generation in high-cold and high-altitude area: The case of Lhasa, Tibet.

The ecological environment in high-cold and high-altitude area is fragile and sensitive, which raise higher claim for municipal solid waste (MSW) management. In the high-cold and high-altitude area, there are problems, such as the mismatch between the actual amount of MSW generated and the scale of transportation and treatment facilities, and the inefficiency of MSW management. In terms of MSW forecasting methods, it is also difficult to forecast due to the lack of data. This study is the first to propose a system dynamics-based method for predicting the amount of MSW generated in high-cold and high-altitude area, and apply it to Lhasa. The research results show that the total amount of MSW generated in Lhasa is small, but the growth rate is fast. Through dynamic simulation, it is found that the synergistic consideration of gross domestic product (GDP) growth rate, urban construction policy and tourism development policy can significantly reduce the growth trend (14% emission reduction in 2030). In addition, strengthening supervision and restraint, publicity and education in high-cold and high-altitude area can produce better waste sorting effects, minimise the pressure on treatment facilities, and improve resource utilisation. Finally, the policy implications are suggested, for example, in the process of MSW management, the impact of economy, urbanisation, tourism and so on, should be taken into account and comprehensively adjusted. It is anticipated that this model and policy implications can be applied to other high-cold and high-altitude cities to provide data support and policy reference for the whole-process management of MSW.

[Effects of Xiangqin Jiere Granules on lipid metabolism and chronic inflammation in different obesity model mice].

This paper aims to study the effect of Xiangqin Jiere Granules(XQ) on lipid metabolism and chronic inflammation in different obesity model mice. The monosodium glutamate(MSG) obese mouse model was established by subcutaneous injection of MSG in newborn mice, and the high fat diet(HFD) obese mouse model was established by feeding adult mice with HFD. The normal mice were assigned into the control group; the MSG obese mice were assigned into MSG model group, XQ4.5 group(Xiangqin Jiere Granu-les, 4.5 g·kg~(-1)), XQ22.5 group(Xiangqin Jiere Granules, 22.5 g·kg~(-1)); the HFD obese mice were assigned into HFD model group, XQ4.5 group, and XQ22.5 group. The mice were intragastrically administrated with saline or XQ for 5 weeks. After that, the body weight, visceral fat mass, liver and thymus weight, and the organ indexes in each group were measured. The levels of triglyceride(TG), total cholesterol(TC), and low-density lipoprotein cholesterol(LDL-c) in serum and liver tissue were detected by the kits. The mRNA expression levels of acetyl CoA carboxylase 1(ACC1), fatty acid synthetase(FAS), diacylgycerol acyltransferase 1(DGAT1) and hepatic lipase(HTGL) involved in lipid metabolism in mouse liver tissue were detected by quantitative real-time PCR(qPCR). The protein levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in serum were detected by ELISA, and the mRNA levels of TNF-α and IL-6 in liver tissue were detected by qPCR. Compared with the control group, MSG and HFD mice showed increased body weight, abdominal circumference, Lee index and visceral fat mass as well as elevated levels of TG, TC, and LDL-c in serum. The model mice had up-regulated gene levels of ACC1, FAS and DGAT1 while down-regulated gene level of HTGL in the liver. Furthermore, the mRNA and protein levels of IL-6 increased in the model mice. Compared with the model mice, XQ treatment decreased the body weight, abdominal circumference, Lee index, and visceral fat mass, lowered the levels of TG, TC, and LDL-c in se-rum, down-regulated the gene levels of ACC1, FAS, and DGAT1 in liver tissue, up-regulated the gene level of HTGL, and down-regulated the mRNA and protein levels of IL-6. To sum up, XQ has good therapeutic effect on different obesity model mice. It can improve lipid metabolism and reduce fat accumulation in obese mice by regulating the enzymes involved in lipid metabolism, and alleviate obesity-related chronic low-grade inflammation.

Jintiange Capsule Alleviates Rheumatoid Arthritis and Reverses Changes of Serum Metabolic Profile in Collagen-Induced Arthritic Rats.

PURPOSE: Jintiange capsule (JTG), an approved drug developed as a substitute for tiger bone (TB), has been clinically applied for osteoporosis therapy since 2003. The drug is composed of bionic TB powder, in which peptides and proteins are primarily enriched from other bone extracts. However, as a precious material of traditional Chinese medicine (TCM), TB has been mainly understood and used in TCM to relieve osteoporosis, rheumatoid arthritis and bone injury. Inspired by those, the purpose of this study was to investigate whether JTG also had an effect on relieving rheumatoid arthritis in collagen-induced arthritic (CIA) rats and explore potential mechanism from the perspective of serum metabolic profile changes. METHODS: JTG was analyzed using Nano LC-MS/MS and orally administered in CIA rats for 6 weeks. After administration, intervention effects of JTG on synovial inflammation, bone micro-architecture and bone metabolism were studied, and the impact of JTG on serum metabolic profiles in CIA rats was investigated by metabolomics. RESULTS: Nine bioactive peptides were identified in JTG. In animal treatments, JTG alleviated paw swelling (P < 0.01), arthritic severity (P < 0.01) and synovial tissue proliferation, as well as inflammatory cell infiltration of ankle joint, decreased bone loss, improved microstructure of bone in CIA rats by regulating bone absorption and formation, specifically increasing bone mineral density (BMD) (P < 0.05), bone volume fraction (BVF) (P < 0.05), trabecular number (Tb.N) (P < 0.05) and decreasing trabecular separation (Tb.Sp) (P < 0.05). Besides, serum IL-6 was down-regulated remarkably in CIA rats (P < 0.05). Furthermore, metabolomics analysis revealed that 32 metabolites were regulated significantly (P < 0.05) by comparison between CIA model and JTG in 360 mg/kg dose. The pathway analysis implied that JTG was involved in regulation of biosynthesis of phenylalanine. CONCLUSION: JTG alleviates rheumatoid arthritis and reverses changes in serum metabolic profile in CIA rats.

Ursolic acid benzaldehyde chalcone, leads to inhibition of cell proliferation and arrests cycle in G1/G0 phase in colon cancer.

The present study investigates the effect of matrine on colon cancer cell viability and apoptosis and tumor growth in mice xenograft model. The results from MTT assay revealed a concentration and time dependent reduction in viability of HCT8 and HT29 colon cancer cells by matrine. The viability of HCT8 and HT29 cells was reduced to 24.67 and 29.32% on treatment with 4 µM/ml concentration of matrine after 48 h (P < 0.05). The results from flow cytometry revealed increase in population of HCT8 and HT29 cells to 77.6 ± 0.3 and 54.0 ± 5.4%, respectively compared to 1.4 ± 0.3 and 2.4 ± 0.7% in control on exposure to 1 µM/ml concentration of matrine. Histone H2AX phosphorylation and expression of Myt1, cyclin A2, cyclin B1 and p53 were increased in HCT8 and HT29 cells on treatment with matrine for 48 h. Matrine treatment also increased the phosphorylation of cdc2 significantly compared to control cells at 48 h (P < 0.05). Results from Annexin-V/FITC-staining showed increase in proportion of apoptotic cells in HCT8 and HT29 cells 67.52 and 68.56 on treatment with 1 µM/ml of matrine. Matrine treatment caused a marked reduction in the growth of HCT8 cell xenograft after 21 days. Thus matrine inhibits cell viability, induces apoptosis and inhibits tumor growth in colon cancer.

Crocetin treatment inhibits proliferation of colon cancer cells through down-regulation of genes involved in the inflammation.

BACKGROUND: The current study was designed to investigate the effect of crocetin on the proliferation inhibition of colon cancer cells and the underlying mechanism. METHODS: MTT assay showed inhibition of proliferation of colon cancer cells in a dose based manner by crocetin treatment. At 30 µM concentration of crocetin proliferation rate of colon cancer cells was reduced to 14% after 24 h. Flow cytometry and fluorescence microscopy revealed induction of apoptosis in colon cancer cells on treatment with crocetin. The tube formation was suppressed significantly in the cultures of HUVEC treated with 30 µM concentration of crocetin compared to the control cultures. RESULTS: The results from transwell assay revealed a significant reduction in the population of DU-145 cells passing through filters of transwell on treatment with crocetin compared to the control cells. Treatment of the DU-145 cells with crocetin caused a significant reduction in the expression levels of NF-κB, VEGF and MMP-9. The results from RT-PCR analysis revealed a significant reduction in the expression of genes involved in inflammation including, HMGB1, IL-6 and IL-8 on treatment of DU-145 cells with crocetin. However, the expression of NAG-1 gene was increased by crocetin treatment in DU-145 cells significantly compared to the control cells. CONCLUSION: Crocetin inhibits growth of colon cancer cells and prevents tube formation through induction of apoptosis. Therefore, crocetin can be used efficiently for the treatment of colon cancer.

Two new chalcones from Shuteria sinensis.

Two new chalcones, 2',3,4,4'-tetrahydroxy-2-prenylchalcone (1) and 3-methoxy-2',4,4'-trihydroxy-2-prenylchalcone (2), together with two known compounds, munsericin (3) and 3,4-dihydroxylonchocarpin (4), were isolated from the ethanol extract of the whole plant of Shuteria sinensis. Their structures were identified by spectroscopic analysis methods, such as 1D and 2D NMR, along with HR-MS data. Glucose metabolism activity of four compounds was tested, compounds 3 and 4 showed effect on the glucose consumption of insulin-resistant HepG2 cells.

mRNA expression and DNA methylation in three key genes involved in caste differentiation in female honeybees (Apis mellifera).

In honeybee (Apis mellifera) colonies, queens and workers are alternative forms of the adult female honeybee that develop from genetically identical zygotes but that depend on differential nourishment. Queens and workers display distinct morphologies, anatomies and behavior, better known as caste differentiation. Despite some basic insights, the exact mechanism responsible for this phenomenon, especially at the molecular level, remains unclear although some progress has been achieved. In this study, we examined mRNA levels of the TOR (target of rapamycin) and Dnmt3 (DNA methyltransferase 3) genes, closely related to caste differentiation in honeybees. We also investigated mRNA expression of the S6K (similar to RPS6-p70-protein kinase) gene linked closely to organismal growth and development in queen and worker larvae (1-day and 3-day old). Last, we investigated the methylation status of these three genes in corresponding castes. We found no difference in mRNA expression for the three genes between 1st instar queen and worker larvae; however, 3rd instar queen larvae had a higher level of TOR mRNA than worker larvae. Methylation levels of all three genes were lower in queen larvae than worker larvae but the differences were not statistically significant. These findings provide basic data for broadening our understanding of caste differentiation in female honeybees.

Evaluation of Helicobacter pylori eradication and drug therapy in patients with functional dyspepsia.

The aim of this study was to assess the effect of Helicobactor pylori (H. pylori) infection and drug therapy on functional dyspepsia (FD) symptoms and gastrointestinal eosinophil count. In this study, 215 continuous FD patients fulfilling Rome III criteria were enrolled. The patients were divided into a H. pylori-positive group and a H. pylori-negative group. The H. pylori-positive group was divided into H. pylori-eradicated and H. pylori-uneradicated groups following H. pylori-eradication treatment, and the H. pylori-negative group was randomly divided into esomeprazole and teprenone treatment groups. The symptom scores of the esomeprazole group were significantly lower compared with those of the teprenone group at week 6 but not at baseline and week 2. Compared with the H. pylori-uneradicated group, eosinophil counts in the antrum and body were significantly reduced in the H. pylori-eradicated group at week 6. The number of gastric eosinophil clusters was significantly higher in the H. pylori-positive group than in the H. pylori-negative group. Eradication was associated with gastric eosinophil counts but did not affect duodenal eosinophil levels. Neither esomeprazole nor teprenone treatments reduced eosinophil levels in the stomach and duodenum of H. pylori-negative patients.

Mitochondrial dysfunction and nuclear-mitochondrial shuttling of TERT are involved in cell proliferation arrest induced by G-quadruplex ligands.

G-quadruplex ligands DODC and TMPyP4 have different binding modes to quadruplex structure and cause cell proliferation arrest. Here we showed that DODC was more efficient in cell growth inhibition than TMPyP4. Both G-quadruplex ligands induced nuclear-cytoplasmic shuttling and accumulation of TERT in mitochondria. This effect was not fully dependent on cellular oxidative stress. DODC induced robust cell apoptosis by perturbing mitochondrial function intensively. Overexpression of TERT could not counteract the effects of DODC on mitochondrial respiratory function. Taken together, our results suggest that interference of mitochondrial function by DODC is one of main targets for its anti-tumor ability.

BRG1 is required for formation of senescence-associated heterochromatin foci induced by oncogenic RAS or BRCA1 loss.

Cellular senescence is an important tumor suppression mechanism. We have previously reported that both oncogene-induced dissociation of BRCA1 from chromatin and BRCA1 knockdown itself drive senescence by promoting formation of senescence-associated heterochromatin foci (SAHF). However, the molecular mechanism by which BRCA1 regulates SAHF formation and senescence is unclear. BRG1 is a chromatin-remodeling factor that interacts with BRCA1 and pRB. Here we show that BRG1 is required for SAHF formation and senescence induced by oncogenic RAS or BRCA1 loss. The interaction between BRG1 and BRCA1 is disrupted during senescence. This correlates with an increased level of chromatin-associated BRG1 in senescent cells. BRG1 knockdown suppresses the formation of SAHF and senescence, while it has no effect on BRCA1 chromatin dissociation induced by oncogenic RAS, indicating that BRG1 functions downstream of BRCA1 chromatin dissociation. Furthermore, BRG1 knockdown inhibits SAHF formation and senescence induced by BRCA1 knockdown. Conversely, BRG1 overexpression drives SAHF formation and senescence in a DNA damage-independent manner. This effect depends upon BRG1's chromatin-remodeling activity as well as the interaction between BRG1 and pRB. Indeed, the interaction between BRG1 and pRB is enhanced during senescence. Chromatin immunoprecipitation analysis revealed that BRG1's association with the human CDKN2A and CDKN1A gene promoters was enhanced during senescence induced by oncogenic RAS or BRCA1 knockdown. Consistently, knockdown of pRB, p21(CIP1), and p16(INK4a), but not p53, suppressed SAHF formation induced by BRG1. Together, these studies reveal the molecular underpinning by which BRG1 acts downstream of BRCA1 to promote SAHF formation and senescence.

Effects of Tai Chi on the protracted abstinence syndrome: a time trial analysis.

While exercise has been shown to reduce the negative effects of substance withdrawal symptoms, no research has investigated if Tai Chi, a traditional Chinese exercise, has similar effects. Here, we observed the physiological effects of Tai Chi on protracted abstinence syndrome (PAS) in female heroin addicts by comprehensively inspecting their immune system function, complete blood count, hepatic function and renal function. To determine the psychological effects, we used the Hamilton Rating Scale for Depression (HRSD) and the rating scale of heroin withdrawal symptoms. We recruited 70 heroin-addicted young women beginning to undergo withdrawal and randomly assigned them into two groups: one group received one-hour Tai Chi exercise every two days (Tai Chi group, n = 36) and the other group did not (control group, n = 34). Thirty-three patients finished this six-month trial. Numerous significant physiological differences were observed between all heroin-addicted subjects (n = 70) and age-matched healthy individuals (n = 18), suggesting a deleterious effect of drug addiction. There were improvements for certain physical parameters between the Tai Chi group (n = 17) and the control group (n = 16), although the differences were not statistically significant. We observed a small significant difference in psychological effects near the 60-day mark between the two groups. Taken together, our results suggest that Tai Chi might have a positive effect on PAS, which future studies can confirm by using an expanded sample size, longer trial time, and more sensitive and specific indicators of psychological and physiological health.

[Discussion on strengthening yin of chinese herbs with bitter-flavor clinical traditional Chinese pharmacology noun terminology standardization research].

Clinical traditional Chinese pharmacology is the subject that study of basic theory of traditional Chinese medicine, property of Chinese materia medica and clinical application. The study on the standardization research of the terminology of clinical traditional Chinese pharmacology is an important premise and foundation to standardization, modernization and internationalization, informationization construction of clinical traditional Chinese pharmacology and is also the important content of the subject construction. To provide some exploring ideas for clinical traditional Chinese pharmacology noun terminology standardization, this article elaborates the concept of strengthening Yin with bitter-flavor herbs in several aspects, such as connotation and the historical origin, the clinical application in the traditional, modern clinic application, and the modern basic research and so on.

Presence of mutation m.14484T>C in a Chinese family with maternally inherited essential hypertension but no expression of LHON.

Essential hypertension (EH, MIM 145500) is the most common cardiovascular disease and affects one-quarter of the world's adult population. Families with EH in a mode of maternal transmission have been occasionally observed in clinical settings and suggested an involvement of mitochondrial DNA (mtDNA) mutation. We aimed to characterize the role of mtDNA mutation in EH. We reported a large Han Chinese family with a maternally inherited EH and an extraordinarily high percentage of sudden death mainly in affected females. Analysis of the entire mtDNA genome of the proband identified a homoplasmic primary mutation m.14484T>C for Leber's hereditary optic neuropathy (LHON), along with several variants indicating haplogroup F1 status. Intriguingly, no maternal member in this family had LHON though they all harbored m.14484T>C. The arterial stiffness of the members carrying mutation m.14484T>C was significantly increased than that of non-maternal members without this mutation. No environmental factor (including age, sex, smoking, diabetes, hyperlipidemia) was correlated with the decreased aortic elastic properties observed in affected members. Mitochondrial respiration rate and membrane potential (ΔΨ(m)) were significantly reduced in lymphoblastoid cell lines established from affected members carrying m.14484T>C when compared to control cell lines (P<0.05). There was an elevation of reactive oxygen species and a compensatory increase of mitochondrial mass in mutant cell lines. Our results suggest that m.14484T>C causes EH under certain circumstance. This study provides a paradigm for diverse phenotypes of the primary LHON mutation and suggests for the necessity of routine cardiac evaluation in patients with the primary LHON mutation.

Is mitochondrial tRNA(phe) variant m.593T>C a synergistically pathogenic mutation in Chinese LHON families with m.11778G>A?

Mitochondrial transfer RNA (mt-tRNA) mutations have been reported to be associated with a variety of diseases. In a previous paper that studied the mtDNA background effect on clinical expression of Leber's hereditary optic neuropathy (LHON) in 182 Chinese families with m.11778G>A, we found a strikingly high frequency (7/182) of m.593T>C in the mitochondrially encoded tRNA phenylalanine (MT-TF) gene in unrelated LHON patients. To determine the potential role of m.593T>C in LHON, we compared the frequency of this variant in 479 LHON patients with m.11778G>A, 843 patients with clinical features of LHON but without the three known primary mutations, and 2374 Han Chinese from the general populations. The frequency of m.593T>C was higher in LHON patients (14/479) than in suspected LHON subjects (12/843) or in general controls (49/2374), but the difference was not statistically significant. The overall penetrance of LHON in families with both m.11778G>A and m.593T>C (44.6%) was also substantially higher than that of families with only m.11778G>A (32.9%) (P = 0.083). Secondary structure prediction of the MT-TF gene with the wild type or m.593T>C showed that this nucleotide change decreases the free energy. Electrophoretic mobility of the MT-TF genes with the wild type or m.593T>C transcribed in vitro further confirmed the change of secondary structure in the presence of this variant. Although our results could suggest a modest synergistic effect of variant m.593T>C on the LHON causing mutation m.11778G>A, the lack of statistical significance probably due to the relatively small sample size analyzed, makes necessary more studies to confirm this effect.

Fast detection of quadruplex structure in DNA by the intrinsic fluorescence of a single-stranded DNA binding protein.

Single-stranded guanine-rich (G-rich) DNA can fold into a four-stranded G-quadruplex structure and such structures are implicated in important biological processes and therapeutic applications. So far, bioinformatic analysis has identified up to several hundred thousand of putative quadruplex sequences in the genome of human and other animal. Given such a large number of sequences, a fast assay would be desired to experimentally verify the structure of these sequences. Here we describe a method that identifies the quadruplex structure by a single-stranded DNA binding protein from a thermoautotrophic archaeon. This protein binds single-stranded DNA in the unfolded, but not in the folded form. Upon binding to DNA, its fluorescence can be quenched by up to 70%. Formation of quadruplex greatly reduces fluorescence quenching in a K+-dependent manner. This structure-dependent quenching provides simple and fast detection of quadruplex in DNA at low concentration without DNA labelling.

G-quadruplex formation in human telomeric (TTAGGG)4 sequence with complementary strand in close vicinity under molecularly crowded condition.

Chromosomes in vertebrates are protected at both ends by telomere DNA composed of tandem (TTAGGG)n repeats. DNA replication produces a blunt-ended leading strand telomere and a lagging strand telomere carrying a single-stranded G-rich overhang at its end. The G-rich strand can form G-quadruplex structure in the presence of K+ or Na+. At present, it is not clear whether quadruplex can form in the double-stranded telomere region where the two complementary strands are constrained in close vicinity and quadruplex formation, if possible, has to compete with the formation of the conventional Watson-Crick duplex. In this work, we studied quadruplex formation in oligonucleotides and double-stranded DNA containing both the G- and C-rich sequences to better mimic the in vivo situation. Under such competitive condition only duplex was observed in dilute solution containing physiological concentration of K+. However, quadruplex could preferentially form and dominate over duplex structure under molecular crowding condition created by PEG as a result of significant quadruplex stabilization and duplex destabilization. This observation suggests quadruplex may potentially form or be induced at the blunt end of a telomere, which may present a possible alternative form of structures at telomere ends.