Restriction Enzymes HpaII Research Articles

First report of a ‘Candidatus Phytoplasma asteris‘‐related strain (16SrI‐B) associated with Morus alba (white mulberry) witches' broom in Iran

White mulberry (Morus alba), belongs to the family Moraceae and is grown domestically and commercially in Iran for use as fresh fruit or dried nuts. During 2018-19, white mulberry plants showing witches' broom (WMWB) symptoms (Fig. 1) were observed sporadically in the Marvast district of Yazd province, Iran. The maximum incidence of disease was 4%. In affected trees, the witches' broom was restricted to some branches and did not affect the whole canopy. Total DNA was extracted from 0.2g of midrib tissue of fresh leaves from a total of 14 diseased plants from five different orchards, and three asymptomatic plants. DNA samples were tested for the presence of phytoplasmas by nested PCR, using primers P1/P7 (Deng & Hiruki, 1; Schneider et al., 4) in the first round, and primers R16F2n/R16R2 (Gundersen & Lee, 3) in the nested round. PCR amplicons of 1.8 and 1.25 kb (respectively) were obtained from all diseased plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 amplicons using RsaI, KpnI, HpaII, HaeIII, AluI, MseI, HhaI, TaqI, BfaI and ThaI restriction endonucleases showed RFLP patterns similar to the 16SrI phytoplasma group. The PCR products from three randomly selected WMWB plants were sequenced and showed 100% sequence identity with each other and 99.6% identity with ‘Ca. P. asteris’ (M30790). One R16F2n/R16R2 16S rDNA sequence from the Marvast WMWB strain was submitted to GenBank (Accession No. MT075612). BLAST searches showed that the WMWB phytoplasma shared more than 99% sequence identity with those of ‘Ca. P. asteris’ and related strains. Phylogenetic analysis using Mega7 software confirmed that the phytoplasma strain associated with WMWB clustered within the 16SrI phytoplasma clades closest to ‘Ca. P. asteris’ (M30790) (Fig. 2). Many phytoplasma strains related to ‘Candidatus Phytoplasma aurantifolia’ (from carrot and sunflower), ‘Ca. Phytoplasma australasia’ (from alfalfa, almond, beetroot, marigold, sesame, squash and tamarisk), ‘Ca. Phytoplasma trifolii’ (from alfalfa, aubergine, cucumber and tomato), ‘Ca. Phytoplasma solani’ (from bindweed and grapevine) and ‘Ca. Phytoplasma omanensis’ (from bindweed and peach) have been reported previously in Yazd province. A 16SrI-B related phytoplasma has been reported associated with Eruca sativa (rocket) phyllody in Yazd province (Esmailzadeh Hosseini et al., 2). To our knowledge, this is the first report of a ‘Ca. Phytoplasma asteris’ (16SrI-B) strain associated with witches' broom of Morus alba in Iran.

Report of ‘Candidatus Phytoplasma hispanicum’ (16SrXIII-E) Associated with Cauliflower Stunt in São Paulo State, Brazil, and Balclutha hebe as Its Potential Vector

A cauliflower stunt disease associated with phytoplasmas occurs in crops of cauliflower (Brassica oleracea var. botrytis) in Sao Paulo state (SP), southeastern region of Brazil. The symptoms are characterized by stunting, extensive malformations of floral parts, leaf reddening, and vessel necrosis. 16SrIII-J and 16SrXV-A phytoplasmas have already been reported in association with this disease in this region (Canale and Bedendo 2013; Rappussi et al. 2012). Herein we report the involvement of 16SrXIII-E phytoplasma with cauliflower stunt disease and the presence of this bacteria in Balclutha hebe leafhopper. DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from leaf veins of seven symptomatic and seven asymptomatic cauliflower samples collected in May 2008 from a farm located in Morungaba (SP, –22.882603, –46.755783). In addition, in an attempt to identify a possible vector for the phytoplasma, insects were captured using a sweep net from a cauliflower farm in Sorocaba (SP, –23.391314, –47.507474), where the disease occurred in high incidence levels (Rappussi et al. 2012). The most abundant leafhopper species collected with the net was identified as B. hebe Kirkaldy, 1900 (Hemiptera: Cicadellidae). DNA was also obtained from whole insects using the same extraction kit as we used for plants. Phytoplasma detection was performed with a nested-PCR assay using P1/Tint and R16F2n/R16R2 universal primers specific to the phytoplasma 16S rRNA gene (Lee et al. 1998; Smart et al. 1996). PCR products of the expected size (∼1.2 kb) were obtained from all of the symptomatic cauliflower but not from the collected symptomless samples. We amplified phytoplasma DNA from four out of 15 B. hebe specimens. Two plant amplicons and two leafhopper amplicons were sequenced (Macrogen, Seoul, Korea). Nucleotide sequence alignment revealed that 16S rDNA of the phytoplasma from cauliflower and insect samples shared ˃99% sequence similarity with Papaya apical curl necrosis phytoplasma, a member of the 16SrXIII-E subgroup and previously described by our group (Melo et al. 2013). Restriction fragment length polymorphism analysis using AluI, RsaI, KpnI, HpaII, MseI, HhaI, MboI, and BstUI restriction enzymes and in silico analysis using the iPhyClassifier online tool (Zhao et al. 2009) also allowed confirmation of the identification of the phytoplasma affecting cauliflower plant and leafhopper samples as member of the 16SrXIII-E subgroup (F = 1.00). A phylogenetic tree was generated with sequences of 16S rDNA of distinct subgroups affiliated with the 16SrXIII group, using the neighbor-joining method and 1,000 bootstrap replicates (MEGA software, Tamura et al. 2011), using Acholeplasma laidlawii as an outgroup. The phylogenetic analysis supported the identification of cauliflower and B. hebe phytoplasmas reported in this study as members of 16SrXIII-E subgroup. Sequences of the 16SrXIII-E phytoplasmas were deposited in GenBank as the accession JN818844 (Cauliflower stunt phytoplasma) and JN818843 (B. hebe phytoplasma). The 16SrXIII phytoplasma group (‘Candidatus Phytoplasma hispanicum’) is diverse and apparently restricted to the American continent, affecting a wide range of plant hosts (Davis et al. 2016). B. hebe is a polyphagous generalist species omnipresent in southern and south Brazil. It is reported as a putative vector for Xylella fastidiosa in plum orchards in south Brazil (Hickel et al. 2001). Also, this species was found in grasses within citrus rows in orchards in Sao Paulo, and one specimen was infected with 16SrIX phytoplasma (Marques et al. 2012). B. hebe was abundant within the cauliflower crops in both locations of this study, and its abundance along with our research results indicate that this leafhopper may play a role as a vector for 16SrXIII-E to cauliflower. However, more refined transmission assays employing phytoplasma and insects maintained in a laboratory are needed to prove B. hebe capacity as a phytoplasma vector. Here, we report ‘Ca. P. hispanicum’ strain as one of the associated phytoplasmas with cauliflower stunt disease in Brazil and B. hebe leafhopper as a putative vector.

Identification and molecular characterization of Azotobacter chroococcum and Azotobacter salinestris using ARDRA, REP, ERIC, and BOX.

Azotobacter chroococcum and A. salinestris do not possess significant and distinct morphological and physiological differences and are often mistaken with each other in microbiological research. In this study, 12 isolates of Azotobacter isolated by standard protocol from soils were identified morphologically and physiologically as A. chroococcum. The isolates were more closely investigated for the molecular differentiation and diversity of A. chroococcum and A. salinestris. For this purpose, the ARDRA technique including HpaII, RsaI, and AluI restriction enzymes, and REP, ERIC, and BOX markers were used. The nifD and nifH genes were also utilized to evaluate the molecular identification of these two species. The 16S rDNA evaluation showed that only four out of the 12 isolates were identified as A. chroococcum and the rest were A. salinestris. The results revealed that HpaII was able to differentiate A. chroococcum from A. salinestris whereas RsaI and AluI were not able to separate them. Moreover, BOX and REP markers were able to differentiate between A. chroococcum and A. salinestris. However, ERIC marker and nifD and nifH genes were unable to separate these species. According to the results, HpaII restriction enzyme is suggested to save time and cost. BOX and REP markers are recommended for differentiation and clear discrimination not only between A. chroococcum and A. salinestris but also among their strains.

Incidence and molecular characterization of a 16SrI-B phytoplasma strain associated with Vitis vinifera leaf yellowing and reddening in the west of Iran

Leaf yellowing and reddening symptoms on Vitis vinifera vines were observed in different grapevine nurseries in the province of Kurdistan in the west of Iran during 2016–2017. Total DNA was extracted from 27 symptomatic and four symptomless plants and tested for the presence of phytoplasma DNA by nested polymerase chain reaction (PCR) using P1/P7 and R16F2n/R16R2 primer pairs. PCR amplicons of about 1.8 and 1.2 kb in size were obtained from all symptomatic V. vinifera but not from symptomless plants. Restriction fragment length polymorphism (RFLP) profiles of R16F2n/R2 amplicons using AluI, HhaI, HpaII, RsaI, TaqI, KpnI, HaeIII and Sau3AI restriction enzymes were identical to each other and to those obtained from 16SrI phytoplasmas, indicating that the phytoplasmas associated with grapevine yellowing and reddening were members of the 16SrI group. A R16F2n/R16R2 16S rDNA sequence of Kurdistan V. vinifera yellowing and reddening strain (KVvY) was submitted to GenBank as accession number MH638316. The Blast analysis of KVvY showed 100% sequence identity with those of the ‘Candidatus Phytoplasma asteris’-related strains. Phylogenetic analysis confirmed that the phytoplasma associated with KVvY clustered within the 16SrI phytoplasma clade closer to the Onion yellows mild strain (OY-M), a 16SrI-B phytoplasma. Restriction analysis using iPhyClassifier confirmed that virtual RFLP patterns of the KVvY were identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession AP006628).

Electrochemical determination of the activity of DNA methyltransferase based on the methyl binding domain protein and a customized modular detector.

An electrochemical method is described for the determination of the activity of the DNA methyltransferase (MTase). The assay was based on the use of a commercially available customized electromagnetic modular detector, which consisted of a magnetic switch, electrical connectors and a screen-printed electrode modified with graphene oxide. The biotinylated single-strand DNA (ss-DNA) S1 was absorbed by streptavidin-modified magnetic beads (MBs) via streptavidin-biotin interaction. The biotinylated ss-DNA S1 was hybridized with the complementary ss-DNA S2. After the symmetrical sequences 5'-CCGG-3' of the duplex DNA (ds-DNA) were methylated by M. SssI CpG methyltransferase (M. SssI MTase), the symmetrical sequences 5'-CCGG-3' in the ds-DNA were recognized by glutathione S-transferase (GST) tagged methyl CpG binding protein 2 (MeCP2). The unmethylated 5'-CCGG-3' sequences were specifically cleaved by HpaII restriction endonuclease. After magnetic separation and washing, HRP-labeled GST tag monoclonal antibody and H2O2 were used as a tracer label and enzyme substrate, respectively. Electrochemical measurement was carried out atpH7.4in the presence of 50μM thionine and 0.5mM H2O2. Stepwise changes in the microscopic features of the SPE surface upon the formation of each layer were studied by scanning electron microscopy. Cyclic voltammetry and differential pulse voltammetry were used to characterize the electrochemical behavior of the different modified electrodes. Under the optimal conditions, the activity of M. SssI MTase can be determined in the activity range of 0.5-125 unit·mL-1 with a detection limit of 0.2 unit·mL-1 (at an S/N ratio of 3). The sensitivity of the immunoassay is 0.489μA·μM-1·cm-2. Graphical abstract Schematic presentation of the electrochemical immunosensor for the determination of the activity of M. SssI CpG methyltransferase (M. SssI MTase). It is based on an electromagnetic modular detector and the use ofglutathione S-transferase tagged methyl CpG binding protein 2 (GST-MeCP2).

Short telomere subtelomeric hypomethylation is associated with telomere attrition in elderly diabetic patients 1.

Telomere shortening is well known to be associated with the aging process and aging-associated diseases, including diabetes. The telomere length and subtelomeric methylation status in peripheral leucocytes (LTL) were compared in elderly type 2 diabetes (T2D) patients and diabetes-free controls (C). The methylation status was analyzed between MspI-TRF lengths and HpaII-TRF lengths by using methylation-sensitive and -insensitive restriction enzyme isoschizomers, MspI and HpaII, respectively. The mean telomere lengths, MspI-TRF or HpaII-TRF, were not significantly different between C and T2D patients. The percentage of fractionated densitometry showed that long and middle telomeres (>9.4 kb, 4.4-9.4 kb) were unaltered but short telomeres (<4.4 kb) in T2D patients were increased compared with C group. The methylation status revealed subtelomeric hypomethylation in short telomeres of T2D patients. When some patients with T2D were treated with 3-hydroxy-3-methylglutaril coenzyme A (HMG-CoA) reductase inhibitors (statin), results seen in short telomere of T2D patients were not observed and were not different from C. This suggested that this altered subtelomeric hypomethylation may be associated with the accelerated telomere shortening in elderly diabetic patients. These results also mean that the subtelomeric hypomethylation can also be influenced by statin treatment in T2D.

Manganese oxide nanoparticles induce genotoxicity and DNA hypomethylation in the moss Physcomitrella patens

The genotoxicity of nanoparticles is a major concern for nano-safety appraisal in the bryophytes as they are the primary colonizers of bare land, indicators of atmospheric pollution and excellent accumulators of trace metals. The present study for the first time evince the in planta genotoxicity of MnONP in Physcomitrella patens a model plant system utilized for evolutionary developmental genetics. The induction of DNA strand breaks was confirmed by comet assay at all tested concentrations corroborated with the enhanced generation of ROS, increase in Mn dissolution, uptake and internalization.Genotoxicity is often coupled with epigenetic alterations. In the present study, global DNA methylation pattern at the level of single cells was studied by the methylation sensitive comet assay using the isochizomeric restriction endonucleases HpaII (digests unmethylated and hemimethylated DNA) and MspI (digests methylated DNA at 5′-CmCGG-3′). MnONP incited DNA hypomethylation in P. patens gametophores treated with the highest concentration of MnONP (20 μg/mL). The DNA hypomethylation incurred upon MnONP exposure was comparable with that of the DNA methylation blocker 5-azacytidine. This can be ascribed to its clastogenic potential mediated by the formation of H2O2, OH and O2¯.There are no reports on the epigenotoxicity of nanomaterials in plants utilizing the detection of DNA damage and DNA methylation. This can open up new avenues of research on the assessment of the epigenotoxic impact of environmentally relevant nanoparticles using bryophytes as model indicator plant system.

Photoelectrochemical determination of the activity of M.SssI methyltransferase, and a method for inhibitor screening.

A photoelectrochemical (PEC) method is describedfor the determination ofthe activity of M.SssI methyltransferase (MTase). The assay relies on enzyme-linkage reactions and a DNA intercalator Ru(bpy)2(dppz)2+ (where bpy is 2,2'-bipyridine, and dppz is dipyrido[3,2-a:2',3'-c]phenazine) which both serves as a PEC signal. The PEC electrode was obtained by immobilizing 5'-amino modified DNA strands (containing the methylation recognition site 5'-CCGG-3') on a polyethylenimine (PEI) coated ITO/SnO2 electrode with glutaraldehyde as crosslinking agent. In the presence of MTase and S-adenosyl-L-methionine, the 5'-CCGG-3' sequence in the DNA on the electrode is methylated. This protects the DNA strands from the shear of the methylation-sensitive restriction endonuclease HpaII. Consequently, more intact DNA strands remain on the surface of the electrode, providing more sites for Ru(bpy)2(dppz)2+ binding which in turn results in a high PEC response. The result demonstrates that the photocurrent increases linearly with the activity of MTase from 5 to 80U·mL-1, and the limit of detection is 0.45U·mL-1. The other MTases does not enhance the photocurrent, suggesting good selectivity of the assay. The method was also applied to rapid evaluate and screen the inhibitors of MTase. This strategy can be utilized to determinate the activity of other DNA MTases with specific DNA sequence. Graphical abstract Schematic presentation of a photoelectrochemical assay based on enzyme-linkage reactions and a photo electrochemical probe combined with the oxalic acid involved cyclic amplification system for the determination of methyltransferase activity.

Mitochondrial DNA G15927A and G15928A variations in patients with multiple sclerosis

BackgroundModern genetics has offered a fresh perspective on the pathology of Multiple Sclerosis (MS). As mitochondrial DNA (mtDNA) variations are held to be potential contributors to the complex pathobiology of MS, the present study tests the claim that mtDNA G15927A or G15928A variations, or both, are associated with MS in an Iranian population. Materials and methodsFollowing DNA extraction from blood samples of 100 subjects with relapsing-remitting MS, and 100 healthy unrelated control subjects, PCR-RFLP analyses was carried out by HpaII restriction enzyme reaction. Electrophoresis was then performed with 3% Agarose gel. As the restriction enzyme did not differentiate between two neighboring nucleotide positions (G15927A and G15928A), all PCR products with a variant allele were sequenced to determine the exact position of the variation. ResultsThe MtDNA G15927A or G15928A variations were observed in 11 of all 100 cases of MS (11%) and in 7 of 100 healthy control subjects (7%) (P = 0.3, OR = 1.6, 95% CI = 0.5–5.2). Having sequenced all the PCR products with the variant allele (11 cases and 7 controls), the mtDNA G15927A variation was found in one of the 100 cases (1%) and 3 of 100 controls (3%) (P = 0.3, OR = 0.3, 95% CI = 0.0–4.1). Therefore, the mtDNA G15928A variation was present in 10 of the 100 cases (10%) and in 4 of 100 controls (4%) (P = 0.09, OR = 2.6, 95% CI = 0.7–12.0). ConclusionNeither mtDNA variation, G15927A or G15928A, was associated with MS in the studied Iranian population. There was a non-significant association of the G15927A and the G15928A variations separately with MS.

Oligonucleotide-modulated photocurrent enhancement of a tetracationic porphyrin for label-free homogeneous photoelectrochemical biosensing

This work reports the first demonstration of an oligonucleotide-modulated label-free homogeneous photoelectrochemical (PEC) biosensing platform based on the adsorption of tetracationic porphyrin (denoted as TMPyP here) onto 1-naphthalenesulfonate anion (NS−)-grafted indium tin oxide electrode (denoted as TMPyP-NS−-ITO), which generates a stable and rapid photocurrent response. We found that when NS−-ITO electrode was subjected to single-stranded oligonucleotide (ssON) before TMPyP adsorption, a remarkable enhancement of photocurrent intensity was observed from the resulted TMPyP-ssON-NS−-ITO electrode with high specificity towards oligonucleotide. A series of investigations were carried out to understand the mechanism of this oligonucleotide-modulated photocurrent enhancement phenomenon. Moreover, the studies of this robust photocurrent enhancement mechanism was successfully extended to develop a signal-on homogeneous PEC biosensing platform for, as a proof-of-concept, label-free M.SssI methyltransferase activity analysis through a judiciously and compatibly engineered signal transduction strategy consisted of hairpin-shaped oligonucleotide probe, restriction endonuclease HpaII, and Exonuclease I. The rationally designed homogeneous PEC biosensor exhibit sensitive PEC response toward M.SssI methyltransferase with a low detection limit of 3.5 mU/mL and a wide linear range from 0.01 to 120 U/mL. Additionally, we show that our homogeneous PEC biosensing platform can be also utilized to screen methyltransferase inhibitors. Therefore, this work will provide a distinctive paradigm for versatile homogeneous PEC biosensing platform that can be used as potential powerful tool toward innovative label-free bioanalytical purposes.

A simple and universal electrochemical assay for sensitive detection of DNA methylation, methyltransferase activity and screening of inhibitors

Many studies have confirmed that DNA methylation is highly correlated with the occurrence and development of various diseases including cancers. In this work, we developed a simple, sensitive, selective, and universal electrochemical biosensor for detection of DNA methylation and assay of DNA methyltransferase (MTase) activity using M.SssI MTase as an example. The thiolated single-stranded DNA S1 was self-assembled on the surface of gold nanoparticles deposition modified glassy carbon electrode via AuS bonding, then hybridization between the DNA S1 and its complementary DNA S2 formed a double-stranded target sequence for both M.SssI MTase and restriction endonuclease HpaII. HpaII could not cleave the target sequence after it was methylated by M.SssI MTase, while the sequence without methylation could be cleaved. Here, we used methylene blue (MB) as electrochemical indicator. The electrochemical signal of MB increased linearly with increasing M.SssI MTase concentration from 0.5 to 25 U/mL and from 25 to 400 U/mL with a detection limit of 0.04 U/mL. Moreover, screening of M.SssI MTase inhibitors 5-azacytidine (5-Aza) and 5-Aza-2′-deoxycytidine (5-Aza-dC) were successfully investigated using the fabricated electrochemical biosensor and showed that the two classic drugs could both inhibit the M.SssI MTase activity with the IC50 of 2.8 μM and 0.37 μM, respectively, indicating potential application in discovery of new anticancer drugs.

Methylation of progesterone receptor isoform A and B promoters in the reproductive system of cows.

The aim of this study was to investigate whether the promoters of progesterone receptor isoform A (PGRA) and B (PGRB) are methylated and to determine the percentage of methylation occurring for each isoform. Genomic DNA was isolated from the corpora lutea (CL) and endometrial slices from cows on Days 2-5, 6-10, 11-16 and 17-20 of the oestrous cycle. DNA was bisulphite-converted and amplified using methyl-specific polymerase chain reaction (PCR) with primers that detect both methylated and unmethylated sequences. The determination of the percentage of the methylation was performed using HpaII and MspI restriction enzymes. Methyl-specific PCR showed partial methylation of PGRA and PGRB promoters in the CL and endometrium during the oestrous cycle. Methylation for PGRA was between 15 and 17% and for PGRB was in the range of 6 to 7.7% during the oestrous cycle in the CL. In the endometrium, the methylation for PGRA was between 6 and 7.3% and for PGRB was between 3 and 4.8% during the oestrous cycle. The data obtained indicate that the higher promoter methylation of the PGRA isoform could be a mechanism for regulation of PGRA inhibitory activity against PGRB and, in this way, methylation may influence the regulation of progesterone action in the CL and endometrium.

Detection of Russian olive witches’-broom disease and its insect vector in Northwestern Iran

Abstract Recently, Russian olive trees showing witches’-broom and little leaf symptoms have been widely observed in northwestern and central Iran. Polymerase chain reaction (PCR) and nested PCR assays using phytoplasma universal primer pairs confirmed phytoplasma symptomatic infection of trees. Sequence analyses showed that ‘Candidatus Phytoplasma asteris’ was the causal agent of the disease in these regions. However, RFLP results using restriction enzymes HpaII, EcoRI, HinfI and AluI indicated that the collected isolates in these regions are genetically different. In addition, leafhopper Macropsis infuscata was recognized as a possible insect vector of the disease for the first time.

Differential methylation of lncRNA KCNQ1OT1 promoter polymorphism was associated with symptomatic cardiac long QT.

To investigate whether the differential methylation of KCNQ1OT1 was associated with the risk of symptomatic long QTc. We investigated the methylation status of KCNQ1OT1 in a cohort of patients (n = 131) with a symptomatic prolonged QTc. All the patients were genotyped for a common promoter polymorphism (rs11023840). They were also genotyped for DNA digested with the methylation-sensitive HpaII restriction enzyme. We found a significant higher frequency of AA genotype (p = 0.02) in the patients compared with healthy controls (n = 240). In the HpaII-digested samples there was a higher frequency of the A-allele among the patients compared with the controls (p = 0.02). Our findings supported a role for the differential methylation/imprinting of KCNQ1OT1 in the risk for symptomatic prolonged QTc.

A label-free electrochemical biosensor for methyltransferase activity detection and inhibitor screening based on graphene quantum dot and enzyme-catalyzed reaction

A label-free electrochemical biosensor for methyltransferase (M.SssI MTase) activity detection and inhibitor screening was developed based on the amplification of graphene quantum dot (GQD) and enzyme-catalyzed reaction. In the construction of the electrochemical biosensor, auxiliary DNA hybridized with capture DNA to form the double-stranded DNA structure which contained a specific recognition sequence (5′-CCGG-3′) for both M.SssI MTase and restriction endonuclease HpaII. The presence of M.SssI MTase caused the methylation of CpG site in the ds-DNA, which could not be digested by HpaII, causing GQD bound to the undigested ds-DNA and acted as new platform for the immobilization of horseradish peroxidase (HRP) through noncovalant assembly. The modified HRP catalyzed the hydrogen peroxide-mediated oxidation of 3, 3′, 5, 5′-tetramethylbenzidine, resulting an electrochemical signal output. The proposed biosensor realized sensitivity detection of M.SssI MTase activity in the range of 1–40UmL−1 with a detection limit of 0.3UmL−1, and this dual-amplified biosensor had been successfully applied in M.SssI MTase activity inhibitor screening of procaine and epicatechin. Furthermore, this proposed strategy can also be extended for other methyltransferase detection, which had a promising application in clinical diagnosis and drug development.

Absolute quantification of DNA methylation using microfluidic chip-based digital PCR

Hypermethylation of CpG islands in the promoter region of many tumor suppressor genes downregulates their expression and in a result promotes tumorigenesis. Therefore, detection of DNA methylation status is a convenient diagnostic tool for cancer detection. Here, we reported a novel method for the integrative detection of methylation by the microfluidic chip-based digital PCR. This method relies on methylation-sensitive restriction enzyme HpaII, which cleaves the unmethylated DNA strands while keeping the methylated ones intact. After HpaII treatment, the DNA methylation level is determined quantitatively by the microfluidic chip-based digital PCR with the lower limit of detection equal to 0.52%. To validate the applicability of this method, promoter methylation of two tumor suppressor genes (PCDHGB6 and HOXA9) was tested in 10 samples of early stage lung adenocarcinoma and their adjacent non-tumorous tissues. The consistency was observed in the analysis of these samples using our method and a conventional bisulfite pyrosequencing. Combining high sensitivity and low cost, the microfluidic chip-based digital PCR method might provide a promising alternative for the detection of DNA methylation and early diagnosis of epigenetics-related diseases.

Incidence and molecular characterization of a 16SrI-B phytoplasma strain associated with Eruca sativa phyllody in Iran

During 2012–2014 surveys for phytoplasma diseases, a phyllody symptomatology was observed in Eruca sativa in Abarkooh (Yazd province, Iran). The highest disease incidence was 32% in Ali Abad-e Shams (Esfandabad), and infected plants showed crown proliferation, witches’ broom, little leaf, flower virescence, phyllody, sterility, and plant stunting. Total DNA extracted from symptomatic and asymptomatic plants was tested for phytoplasma presence verification by direct PCR using P1/P7 primers and nested PCR using P1/P7 and R16F2n/R16R2 primers pairs. PCR amplicons of about 1.8 and 1.25 kb respectively, were obtained from all symptomatic E. sativa plants, but not from the symptomless ones. Restriction fragment length polymorphism analysis of R16F2n/R2 amplicons using AluI, HhaI, HinfI, HpaII, MseI, RsaI and TaqI restriction enzymes showed profile identical to each other and also to those of 16SrI phytoplasmas indicating that the phytoplasmas, associated with E. sativa phyllody are members of 16SrI group. The consensus sequence of Ali Abad-e Shams E. sativa phyllody strain showed 100% identity with those of the ‘Candidatus Phytoplasma asteris’-related strains. Phylogenetic analysis confirmed that this phytoplasma clustered within the 16SrI phytoplasma clade closer to the onion yellows mild strain (OY-M) a 16SrI-B phytoplasma. Restriction analysis using iPhy Classifier confirmed that virtual RFLP patterns of the phytoplasma were identical (similarity coefficient 1.00) to OY-M strain. A 16SrI-B related phytoplasma has been reported associated with rapeseed (Brassica napus) phyllody in Iran and Eruca sativa may be a secondary host for the 16SrI-B phytoplasma associated with rapeseed phyllody.

Analysis of smad4 gene promoter methylation in pancreatic and endometrial cancers

Background. Promoter hypermethylation of the SMAD4 gene has been registered in some cancer types, but in general doesn?t appear to be a frequent event in carcinogenesis. However, only a few published studies deal with this topic and not many cancer types have been analyzed. The aim of this study was to establish SMAD4 gene promoter methylation status in pancreatic and endometrial cancers. Methods. Patients included in the study (62 subjects) were diagnosed and surgically treated at the University of Belgrade, Clinical Center of Serbia. Patients with pancreatic carcinoma (17 subjects) underwent surgical removal of the pancreatic adenocarcinoma at the First Surgical Clinic, while the patients with endometrial carcinoma (45 subjects) underwent hysterectomy with adnexectomy at the Institute for Gynecology and Obstetrics. Extraction of DNA from fresh tissue samples was performed and the methylation status of the SMAD4 gene promoter was studied by a previously designed PCR-based HpaII and MspI restriction enzyme assay. The resulting PCR products were analyzed by electrophoresis in 2% agarose gels. Results. Neither of the analyzed samples was found to be hypermethylated. Conclusion. This is the first report on SMAD4 methylation status in pancreatic and endometrial tumor specimens, and supports the viewpoint that SMAD4 hypermethylation is not a common event in malignant tumors. Nevertheless, promoter hypermethylation remains a candidate mechanism for SMAD4 inactivation in malignant tissue as a potential cause of decreased or lost SMAD4 expression in certain tumor types, and should be further investigated in different tumor types and larger cohorts of patients.

Electrochemical biosensor for hydroxymethylated DNA detection and β-glucosyltransferase activity assay based on enzymatic catalysis triggering signal amplification

A simple and sensitive electrochemical biosensor was fabricated for hydroxymethylated DNA detection and T4 β-glucosyltransferase activity assay based on the glycosylation of hydroxymethylated DNA and HpaII restriction endonuclease system triggering signal amplification. After immobilizing double-stranded DNA (dsDNA) on the surface of carboxyl functionalized Fe3O4 nanoparticle, the glycosylation reaction of hydroxymethylated cytosine can be performed with the catalytic effect of T4 β-glucosyltransferase using uridine diphosphoglucose as glucose donor. Then, 4-mercaptophenylboronic acid (4-MPBA) can be further reacted with glucose and captured on Fe3O4 nanoparticle surface. After the dsDNA was digested by HpaII, the fragment with 4-MPBA can be released from Fe3O4 nanoparticle surface. After magnetic separation, this fragment can be further modified on gold electrode surface through the interaction between Au and −SH, which can result in a decreased electrochemical signal of [Fe(CN)6]3−. According to this detection strategy, the electrochemical biosensor showed high sensitivity with the low detection limits of 0.14nM and 0.27 unit/mL for hydroxymethylated DNA and T4 β-glucosyltransferase. The fabricated biosensor also presented high selectivity and acceptable reproducibility. In addition, the inhibition activity of 4-phenylimidazole towards T4 β-glucosyltransferase was also investigated. This work provides a novel platform for hydroxymethylated DNA detection, T4 β-glucosyltransferase activity assay and its inhibition screening.

Epigenetic Silencing of Catalase Induces Accumulation of Reactive Oxygen Species in Chronic Lymphocytic Leukemia B Cells Leading to Activation of Axl: An Escape Strategy?

Background: A challenge for novel therapeutic strategies will be the fine tuning of intracellular reactive oxygen species (ROS) signaling in order to effectively deprive cells from ROS-induced tumor promoting events and subsequent tipping the balance to ROS-induced apoptotic signaling. ROS plays a critical role in regulation of the pro-survival receptor tyrosine kinase (RTK) signaling pathways in human cancers. Studies have identified mitochondrial metabolism as the key source for abundant ROS in chronic lymphocytic leukemia (CLL). Unlike in other malignant cells, increased oxidative phosphorylation but not increased aerobic glycolysis has been found in CLL B-cells. While we have detected constitutively phosphorylated/active RTKs like Axl in CLL B-cells, the mechanism of activation remains largely undefined. Here we report that (i) elevated ROS activates the Axl signaling pathway in CLL B-cells and, (ii) mechanism of increased ROS accumulation in the leukemic B-cells.Methods: CLL B- and normal B-cells were purified from peripheral blood of previously untreated CLL patients and normal healthy individuals, respectively using a RossetteSep B-Cell enrichment kit. CLL B-cells were exposed to H2O2 for 5 min and cell lysates were analyzed for activation of RTKs including Axl by immunoprecipitation/Western blots. We also examined expression status of SIRT3, acetylated-superoxide dismutase (SOD)2 and catalase in CLL B-cells by Western blots. Catalase mRNA levels in CLL B-/normal B-cells were determined by qRT-PCR. In some experiments, genomic DNA was isolated from CLL B-/normal B-cells for catalase promoter methylation studies. Finally, accumulation of O2- and H2O2 in CLL B- and normal B-cells were measured by flow cytometry after treating the cells withdihydroethidium (DHE) and dichlorohydrofluorescein diacetate (DCFDA), respectively.Results: We found that enforced induction of ROS significantly increases tyrosine phosphorylation levels on multiple cellular proteins in CLL B-cells (Fig. 1). Further analysis finds that increased ROS activates Axl and its downstream targets AKT/Erk1/2 MAPK and the fibroblast growth factor receptor (FGFR) which we recently defined as a downstream target of Axl; while ROS accumulation did not show any significant effect on other RTKs, cMET or IGFR1, in CLL B-cells. Interestingly, the histone deacetylase SIRT3 which activates mitochondrial SOD2 via deacetylation, we found, was overexpressed in CLL B-cells as compared to normal B-cells indicating more efficient conversion of O2- into H2O2 in the leukemic B-cells (Fig. 2). However expression of catalase, which converts H2O2 into O2 and H2O, was reduced significantly in CLL B-cells as compared to normal B-cells both at mRNA and protein levels. To delineate the mechanism of reduced expression of catalase in CLL B-cells we studied the human catalase promoter region for potential methylation sites using the method of HpaII restriction enzyme digestion where HpaII is unable to cut DNA (CCGG site) when the internal cytosine is methylated. Initial findings from this approach suggest that while the catalase gene promoter in CLL B-cells may contain methylated cytosines, the catalase gene in normal B-cells may not confer methylation in the promoter region. Next, genomic DNA from CLL B-cells (n=10) and normal B-cells (n=6) was subjected to bisulfite treatment, followed by PCR amplification and sequencing. This approach revealed no significant promoter methylation of the catalase gene in normal B-cells, while moderate to heavily methylated promoter of the catalase gene was detected in CLL B-cells from majority of CLL patients (9 of 10). Further studies are in progress to examine catalase promoter methylation status in a larger cohort of CLL patients and its association with H2O2 accumulation, RTK phosphorylation and risk-factors. Indeed, flow cytometric analysis finds lower levels of O2- but accumulation of H2O2 in CLL B-cells as compared to normal B-cells. Taken together, these findings suggest that although SOD2 remains highly active, increased accumulation of H2O2 may occur in CLL B-cells due to epigenetic silencing of the catalase gene.Conclusion: These observations may explain, at least in part, the expression of constitutively active Axl signaling pathway in CLL B-cells that plays a role in CLL B cell survival and may account for the prolonged survival and/or apoptotic resistance of the leukemic B-cells. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.