Single PCR Product Research Articles

First report of saffron latent virus in Crocus sativus from Hungary.

In early April of 2018 we sampled asymptomatic autumn flowering Crocus plants (Fig. S1.) in a private collection in Hajdú-Bihar county, Hungary. From each species (Cr. kotschyanus subsp. kotschyanus, Cr. sativus, Cr. speciosus) 200 mg leaf sample was collected from 5 neighboring shoot, which were treated as one sample. ELISA tests were carried out in duplicates using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) on the samples (Agdia, Elkhart, IN, USA). A sample was considered positive if the absorbance was at least three times greater than that of the negative control. Only one sample tested positive; the absorbance values of Cr. sativus leaves were 0.013 and 0.014, while the negative controls were 0.002 and 0.003, respectively. The samples were further tested by RT-PCR for potyviruses (Salamon and Palkovics 2005), tomato spotted wilt virus (TSWV) (Nemes and Salánki 2020) and nepovirus subgroup A (Digiaro et al. 2007). Total nucleic acid was extracted with the phenol-chloroform method of White and Kaper (1989), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) using random hexamer primer. The samples were negative for TSWV and nepovirus subgroup A, but a single PCR product of ~ 1700 nucleotide (nt) was amplified with potyvirus specific primers and cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA). The 1726 nt long insert sequence, including the complete coat protein region was determined and deposited in the NCBI GenBank database (Accession No: OR425160). Digestion of the original PCR products with restriction enzyme SacI yielded only the predicted restriction fragments (364 / 1362 bp), indicating the presence of only a single potyvirus in the infected sample. BLASTn analysis of the CP cistron revealed the highest nt identities to saffron latent virus (SaLV) Iranian isolates (GenBank AccNo.: MN990394 - 85.44%, MN990415 - 85.39% and RefSeq: NC_036802 - 84.05%). For phylogenetic analyses MEGA11 (Tamura et al. 2021) was used. The resulting Maximum Likelihood tree (Fig. S2) showed that all Iranian SaLV isolates grouped together, while the Hungarian isolate is on an adjacent branch, separate from other virus species, and supported with 100% bootstrap values. From these results, it appears that the Hungarian isolate has been separated from the Iranian clade, and has evolved separately as a distinct lineage. We were unable to fulfill Koch's postulates as all available Crocus sativus plants were infected with SaLV. Latent potyvirus infection of Crocus species, by bean yellow mosaic virus (BYMV), iris mild mosaic virus (IMMV), iris severe mosaic virus (ISMV) and turnip mosaic virus (TuMV) has been reported by Grilli Caiola and Faoro (2011). SaLV was first reported from Iran (Parizad et al. 2017), but to our knowledge has never been reported from Europe or from any current EPPO member state. Since Crocus species can be asymptomatic virus reservoirs, it is important that any certification scheme for production should require laboratory tests to prove the health of the plants; or advise growers to keep possible high value susceptible crops such as breeding material and nuclear stocks at a distance from crocuses to mitigate virus transmission between stocks. It is also advisable to grow infected lots far from healthy stocks and protected wild hosts. To our knowledge, this is the first report of SaLV from Hungary and from Europe.

Diversity of Microbial Functional Genes Should Be Considered During the Interpretation of the qPCR Melting Curves.

Soil microorganisms play an essential role in biogeochemical cycles. One approach to study these microbial communities is quantifying functional genes by quantitative PCR (qPCR), in which a melting curve analysis is usually assessed to confirm that a single PCR product is being quantified. However, the high diversity of functional genes in environmental samples could generate more than one peak in those curves, so the presence of two or multiple peaks does not always indicate nonspecific amplification. Here, we analyzed the taxonomic diversity of soil microorganisms harboring functional genes involved in nitrogen (N) and phosphorus (P) cycles, based on a database of genomes and metagenomes, and predicted the melting curve profiles of these genes. These functional genes were spread across many bacterial phyla, but mainly Proteobacteria and Actinobacteria. In general, the melting curves exhibited more than one peak or peaks with shoulders, mainly related to the variation of the nucleotide composition of the genes and the expected size of the amplicons. These results indicate that the melting curves of functional genes from environmental samples should be carefully evaluated, being in silico analyses a cost-effective way to identify inherent sequence diversity and avoid interpreting multiple peaks always as unspecific amplifications.

MOLECULAR CHARACTERIZATION AND ANTIMICROBIAL SUSCEPTIBILITY OF MRSA ISOLATED FROM CHRONIC HEMODIALYSIS OUTPATIENTS AND THEIR CORRELATION TO MRSA COLONIZATION AMONG HEALTHCARE WORKERS

The carriage of methicillin-resistant Staphylococcus aureus (MRSA) among dialysis patients is remarkable not only in terms of the risks of developing infections, but also in playing a principle part in transmission among dialysis unit staff. The aim of this study was to detect the colonization of Methicillin-sensitive Staphylococcus aureus and MRSA carriage. Also, our aim was to determine the relatedness of MRSA isolates and the potential routes of transmission using PCR- Restriction Fragment Length Polymorphism (PCR-RFLP) in Hemodialysis Unit of El Zagazig General Hospital, a tertiary medical center in Sharqia, Egypt. This study was conducted on 150 chronic hemodialysis outpatients and 200 non clinical control samples including environmental and healthcare workers (HCWs). Antibiotic susceptibility by VITEK-2 and disc diffusion, PCR amplification of mecA, pvl and coa genes and RFLP-PCR were conducted during the study period. In this study 3.3% of the patients and 3.2% of HCWs colonized with pvl positive MRSA. Fifty percent of MRSA isolates showed a single band PCR product amplification of 810bp fragment corresponding to coa gene. Ten distinct MRSA RFLP banding patterns designated as H1-H10 were obtained. The majority of strains belonged to RFLP banding pattern H1 (33.33%).The prevalence of MRSA carriage among hemodialysis patients was 14% and 9.7 % among HCWs with similar polymorphism patterns. The presence of one major coa gene type confirmed the occurrence of hospital acquired-associated MRSA.

Leucocin C-607, a Novel Bacteriocin from the Multiple-Bacteriocin-Producing Leuconostoc pseudomesenteroides 607 Isolated from Persimmon.

Leuconostoc pseudomesenteroides 607, isolated from persimmon fruit, was found to have high inhibitory activity against Listeria monocytogenes and several other Gram-positive bacteria. Inhibitory substances were purified from culture supernatant by ion-exchange chromatography, Sep-Pak C18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Two antibacterial peptides were observed during the purification procedures. One of these peptides had a molecular size of 4623.05Da and a partial N-terminal amino acid sequence of NH2-KNYGNGVHxTKKGxS, in which the YGNGV motif is specific for class IIa bacteriocins. A BLAST search revealed that this bacteriocin was similar to leucocin C from Leuconostoc mesenteroides. Leucocin C-specific primers were designed and a single PCR product was amplified. Analysis of the nucleotide sequence has revealed a putative peptide differing by only one amino acid residue from the sequence of leucocin C. No identical peptide or protein has been reported in the literature, and this peptide, termed leucocin C-607, was therefore considered to be a new variant of leucocin C produced by Leuc. pseudomesenteroides 607. Another antibacterial peptide purified from the same culture supernatant had a molecular size of 3007.7 or 3121.97Da. However, detailed information regarding this second peptide remains to be determined. Distinct characteristics, such as heat stability and inhibitory spectrum, were observed for the two bacteriocins produced by Leuc. pseudomesenteroides 607. These results suggested that Leuc. pseudomesenteroides 607 produces leucocin C-607 along with another unknown bacteriocin.

High-Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science.

Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high-resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual-dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics.

Single Genome Sequencing of Expressed and Proviral HIV-1 Envelope Glycoprotein 120 (gp120) and nef Genes.

The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death ( Lamers et al., 2016a and 2016b; Rose et al., 2016 ). This method optimizes protocols from previous publications ( Palmer et al., 2005 ; Norström et al., 2012 ; Lamers et al., 2015 ; 2016a and 2016b; Rife et al., 2016 ) to produce single distinct PCR products that can be directly sequenced and includes several cost-saving and time-efficient modifications.

Involvement of Allele-Specific Methylation of Asparagine Synthetase Gene in Asparaginase Sensitivity of BCP-ALL

Asparaginase is one of the most important components for the treatment of ALL. ALL cells are supposed to be unable to synthesize adequate amounts of Asparagine (Asn), and, therefore, depend on extracellular source of Asn to survive. Asparaginase therapy induces the depletion of serum Asn by catalyzing the deamination of Asn and leads to cell death of ALL cells. Asparagine synthetase (ASNS) is an enzyme that produces Asn from Aspartic acid. Thus, silencing of the ASNS gene in ALL cells could be crucial for complete starving ALL cells of the Asn. Considering that the ASNS gene has a CpG island in its promotor, aberrant methylation of CpG island could be one of epigenetic mechanisms for silencing of ASNS gene in ALL cells. Previous qualitative analysis of ALL samples using methylation-specific restriction enzyme revealed frequent methylation of CpG island in the ASNS gene. However, associations of methylation status of ASNS gene with its expression level and sensitivity to asparaginase in ALL cells remain unknown. Moreover, little is known about mechanisms for leukemia-specific ASNS gene silencing by methylation. To shed light on these issues, we analyzed a large panel of BCP-ALL cell lines. We quantified ASNS gene expression level by real time RT-PCR in 79 BCP-ALL cell lines cultured in the presence or the absence of L-asparaginase (L-asp), and determined IC50 values of L-asp using alamar blue assay. In the majority of cell lines, although degree of the induction was highly variable, ASNS gene expression level was upregulated in the presence of L-asp. IC50 value of L-asp showed significant correlation with ASNS gene expression level cultured in the presence of L-asp (r=0.222, p=0.049) rather than that in the absence of L-asp (r=0.193, p=0.089). We next analyzed methylation status of the ASNS gene in 79 BCP-ALL cell lines by bisulfite PCR sequencing using a next-generation sequencer (NGS). Strong correlation was confirmed between mean % methylation by NGS and Sanger sequencing in representative cell lines. Of importance, mean % methylation in 79 BCP-ALL cell lines showed significant negative correlation with ASNS gene expression level cultured in the presence of L-asp (r=-0.482, p=6.73x10-6) and, subsequently, IC50 value of L-asp (r=-0.39, p=3.86x10-4). Unexpectedly, % methylation of 79 cell lines distributed in three clusters; 15 cell lines (19%) were highly methylated (>66%, median; 89%), 26 cell lines (32.9%) were moderately methylated (33-66%, median; 40%), and 38 cell lines (48.1%) were weakly methylated (<33%, median; 3.7%). In the majority of moderately methylated cell lines, histograms of % methylation in each read of NGS showed two peaks of high and low methylation, suggesting an allele-specific methylation. In the middle of CpG island, tandem repeat polymorphism of 14bp nucleotides is located adjacent to methylation-specific restriction enzyme site of Aor13HI. Of note, in 7 out of 8 moderately methylated cell lines with heterozygous tandem repeat genotype, only single PCR product was detectable when PCR was performed after Aor13HI treatment, whereas two PCR products derived from two- and three-repeat alleles was detectable when PCR was performed without treatment, indicating an allele-specific methylation. We next analyzed a possible one-allele-loss of the ASNS gene in highly methylated (>66%; 8 cell lines) and weakly methylated (<20%; 12 cell lines) cell lines. We directly sequenced genotype in a portion of introns 2 and 4 and exon 5 based on the imputated SNP genotypes, and confirmed heterozygous genotype in every cell lines at least in one of eight SNPs analyzed, demonstrating that loss-of-heterozygosity is not the mechanism for high or low methylation of the ASNS gene. Similar pattern of methylation was observed in 52 BCP-ALL samples. Taken together, these observations indicate that stepwise allele-specific methylation of ASNSgene is critically involved in the sensitivity to L-asp of BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.

Occurrence of Blastocystis sp. in water catchments at Malay villages and Aboriginal settlement during wet and dry seasons in Peninsular Malaysia.

In the tropics, there are too few studies on isolation of Blastocystis sp. subtypes from water sources; in addition, there is also an absence of reported studies on the occurrence of Blastocystis sp. subtypes in water during different seasons. Therefore, this study was aimed to determine the occurrence of Blastocystis sp. subtypes in river water and other water sources that drained aboriginal vicinity of highly endemic intestinal parasitic infections during wet and dry seasons. Water samples were collected from six sampling points of Sungai Krau (K1–K6) and a point at Sungai Lompat (K7) and other water sources around the aboriginal villages. The water samples were collected during both seasons, wet and dry seasons. Filtration of the water samples were carried out using a flatbed membrane filtration system. The extracted DNA from concentrated water sediment was subjected to single round polymerase chain reaction and positive PCR products were subjected to sequencing. All samples were also subjected to filtration and cultured on membrane lactose glucuronide agar for the detection of faecal coliforms. During wet season, Blastocystis sp. ST1, ST2 and ST3 were detected in river water samples. Blastocystis sp. ST3 occurrence was sustained in the river water samples during dry season. However Blastocystis sp. ST1 and ST2 were absent during dry season. Water samples collected from various water sources showed contaminations of Blastocystis sp. ST1, ST2, ST3 and ST4, during wet season and Blastocystis sp. ST1, ST3, ST8 and ST10 during dry season. Water collected from all river sampling points during both seasons showed growth of Escherichia coli and Enterobacter aerogenes, indicating faecal contamination. In this study, Blastocystis sp. ST3 is suggested as the most robust and resistant subtype able to survive in any adverse environmental condition. Restriction and control of human and animal faecal contaminations to the river and other water sources shall prevent the transmission of Blastocystis sp. to humans and animals in this aboriginal community.

Hae III locus at Major Histocompatibility Complex (MHC) class II region hints duplicated DQA genes in Indian mithun (Bos frontalis)

In bovines, duplication of major histocompatibility complex (MHC) class II DQ genes increase the advantage of high genetic polymorphism in the region multifold to produce better immune response and population fitness. In this study, the DQA gene duplication was explored at MHC class II locus in mithun (Bos frontalis, Bofr), a unique bovine of North-East region of India. A 776 nucleotide long genomic region encompassing hyper-variable exon 2 of Bofr-DQA was amplified in 79 mithuns and digested with Hae III restriction enzyme for PCR-RFLP analysis. The analysis revealed some of the restriction patterns, which were carrying the total fragment size of the alleles aggregating more than a heterozygous condition. Colony PCR-RFLP of clones of mithun DQA by Hinf I enzyme revealed a total of three DQA alleles in single PCR product. The RFLP of direct PCR and clone (colony) PCR products indicated the amplification of three DQA alleles at a time, suggesting duplication of the DQA locus in mithun. Further, the RFLP based typing of mithun DQA locus revealed the presence of five different DQA1 and DQA2 alleles in different combinations during duplication in mithun population. The duplication of DQA, carrying both DQA1 and DQA2 alleles gene was found to be present in nearly half of the mithun population. In mithun, similar to other bovines, DQA gene duplication may have much importance in creating more and diversified MHC class II molecules, thus conferring an advantage to bind with a more number of pathogenic antigens.

Taxonomic confirmation of mud crab species (genus Scylla) in Bangladesh by nuclear and mitochondrial DNA markers

Taxonomy of mud crabs genus Scylla has been misidentified for several years due to their high morphological plasticity. Several reports concerning mud crab have been published with misleading identification in Bangladesh. In this study, partial fragments of nuclear and mitochondrial DNA of Scylla species obtained from four locations along the Bangladesh coast were used to resolve taxonomical ambiguity of mud crab species. A single PCR product from the nuclear first internal transcribed spacer (ITS-1) marker and phylogenetic trees constructed based on 16S rDNA sequences indicated that all Scylla species obtained in this study were S. olivacea. Both molecular data and morphological characters revealed that S. olivacea is the only major species in Bangladesh coastal waters. Further, the 16S rDNA haplotypes significantly differed with known S. serrata by 33%. From this study it is clear that ‘S. serrata’ commonly reported from Bangladesh should be S. olivacea.

Quantitative detection of four pome fruit viruses in apple trees throughout the year

A one-step real-time RT-PCR assay (RT-qPCR) with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed to detect and quantify RNA targets from Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple chlorotic leaf spot virus (ACLSV) in infected apple trees. Single PCR products of 87 bp (ApMV), 70 bp (ASGV), 104 bp (ASPV) and 148 bp (ACLSV) were obtained, and melting curve analyses revealed distinct melting temperature peaks for each virus. A dilution series using in vitro synthesized transcripts containing the target sequences as standards yielded a reproducible quantitative assay, with a wide dynamic range of detection and low coefficients of variance. The content of selected viruses in apple plant tissues was stable throughout the year, and their accumulation did not significantly change between different plant tissues. The only minor exceptions were for ApMV and ACLSV, in which noticeable differences in their concentrations in various biological material were observed within the year. This divergence did not influence their year-round detectability. This one-step RT-qPCR assay is a valuable tool for year-round diagnostics, and molecular studies of the biology of ApMV, ASGV, ASPV and ACLSV.

Development of INDELs markers in oilseed rape (Brassica napus L.) using re-sequencing data

Insertions/deletions (INDELs), a type of abundant length polymorphisms in the plant genomes, combine the characteristics of both simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNP), and thus can be developed as desired molecular markers for genetic studies and crop breeding. There has been no large-scale characterization of INDELs variations in Brassica napus yet. In this study, we identified a total of 538,691 INDELs in size range of 1–10 bp by aligning whole-genome re-sequencing data of 23 B. napus inbred lines (ILs) to the B. napus genome sequence of ‘Darmor-bzh.’ Of these, 104,190 INDELs were uniquely mapped on the pseudochromosomes of the reference genome. A set of 595 unique INDELs of 2–5 bp in length was selected for experimental validation in the 23 ILs. Of these INDELs, 530 (89.01 %) produced a single PCR product and were single locus. A total of 523 (87.9 %) INDELs were found polymorphic among the 23 ILs. A genetic linkage map containing 108 single-locus INDELs and 89 anchor SSR markers was constructed using 188 recombinant ILs. The majority of INDELs markers on the linkage map showed consistency with the pseudochromosomes of the B. napus cultivar ‘Darmor-bzh.’ The INDELs variations and markers reported here will be valuable resources in future for genetic studies and molecular breeding in oilseed rape.

Molecular Identification of Candida dubliniensis among Candida albicans Isolated from Oral Cavity of Cancer Patients using PCR-RFLP, in a Tertiary Care Hospital in Kashmir, India

Aims: To retrospectively evaluate 186 stock strains of C. albicans strains isolated from oral cavity of HIV negative patients with various malignancies for the presence of C. dubliniensis isolates among them by PCR-RFLP. Place and Duration of Study: Department of Microbiology, Sher-i-Kashmir Institute of Medical Sciences, Srinagar, between October 2013 and October 2014. Methodology: This study included 186 stock strains of C. albicans tentatively identified by phenotypic methods like germ tube formation in human serum, colony color on chromogenic Original Research Article Jan et al.; BMRJ, 14(2): 1-7, 2016; Article no.BMRJ.25465 2 candida differential agar, characteristic morphology on corn meal agar and assimilation of sugars isolated from HIV negative patients with various malignancies. DNA extraction was performed by chemical method. PCR amplification of ITS1-5.8S-ITS2 rDNA region was achieved using the ITS1 and ITS4 primer pairs which amplify the ITS region of both species, providing a single PCR product of expected size (540 bp). There is no visible difference between these two species with regard to their ITS PCR products. Digestion of amplified products was performed by using restriction enzyme BlnI (AvrII) which cleaves DNA where there is a CCTAGG sequence. The products of digestion generate one band of 540 bp for C. albicans, and two bands of 200 bp and 340 bp for C. dubliniensis because BlnI has one recognition site within the ITS region of C. dubliniensis, whereas none within that of C. albicans. Results: Of the 186 isolates tested, no C. dubliniensis was found by PCR-RFLP. Conclusion: Our results of not finding C. dubliniensis in this subset of patients support the need for further investigations into the prevalence of this species among other clinical samples and other susceptible patient populations.

Impact of Finding of Low Level Kinase Domain Mutations Using Ultra Deep Next Generation Sequencing in Patients with Chronic Phase CML

▪Kinase domain (KD) mutations in the BCR-ABL1 gene are associated with resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML). Next-generation Sequencing (NGS) allows detection of low-level KD mutations but its relevance in clinical practice remains debated, and the incidence and prognostic significance of finding of low-level KD mutation in chronic phase (CP) patients is unknown.We analyzed the outcome for 121 newly diagnosed CP-CML patients treated with TKIs (imatinib 111, nilotinib 7 and dasatinib 3) who we routinely screened for KD mutations using ultra-deep NGS regardless of response to TKI. When a mutation was found by ultra-deep NGS (using Illumina NexteraXT and MiSeq platform) all available previous cDNA samples were analyzed to establish the date of its first occurrence and subsequent kinetics. ABL1 transcripts from 27 healthy donors were sequenced and used as a control. Sequencing for both single and nested PCR products were compared, and samples were re-sequenced in two independent runs in order to analyze reproducibility of variants detection.Median age of patients was 56 years and the Sokal risk score was 'low' in 38% patients, 'intermediate' in 15.5% and 'high' in 46.5%.A mutation was detected in 25 of the 121 patients (20.6%) at a median time of 14 months from starting TKI. 19 different mutations were identified, the most frequents being F317L (n=17), Y253H (n=15), M244V (n=14) and T315I (n=10).All mutations previously detected by Sanger sequencing were also found using ultra-deep NGS. In addition low-level mutations (<20%) were found using ultra-deep NGS in 23 samples (16 patients), while not being detected using Sanger sequencing. Multiple mutations were found in 14 samples (10 patients).Eighteen patients discontinued imatinib while still in CP and received dasatinib, nilotinib or an allogeneic stem cell transplantation. The overall progression-free survival (absence of advanced phase) at 5 years was 85%. Complete cytogenetic responses (CCyR) and major molecular responses (MMR) with front-line TKI therapy were achieved in 74 (61%) and 52 (43%) patients respectively.We stratified patients according to response to TKI therapy as per 2009 ELN guidelines (Baccarani et al., 2009): patients with optimal response ('responders', R), failure at any time ('non-responders', NR) and sub-optimal response (SR). Patients with known TKI dose-interruption were analyzed separately. Mutations were classified and analyzed according to clinical relevance (clinically relevant versus clinically non relevant mutations, Branford et al., 2009).Among non-responders (NR), 37% of patients developed a mutation (29% among patients who lost CCyR), compared to only 5% among responders (2 patients, both with KD mutations sensitive to imatinib). Among patients with suboptimal response (SR), 19% developed a mutation while being in CCyR but not in MMR with all clinically relevant mutated clone percentages increasing in patients being treated with a TKI to which the KD mutation was conferring resistance. Similarly in all NR patients the mutated clone percentage rose in sequential samples if a clinically relevant mutation was detected.Patients harboring a mutant clone had a poorer PFS at 5 years compared to patients without mutation (67% versus 92%, p<.001). By multivariate analysis, factors associated with worse EFS (progression to AP/BP, death or loss of CCyR) were the presence of a KD mutation and failure to achieve CCyR at 12 months (relative risks 5.4 and 3.6, p=.003 and p=.015, respectively).We next evaluated the incidence and impact of finding of KD mutations at the early molecular response time point (3-months BCR-ABL1 transcript level). Samples from 41 patients were analyzed at 3 months of which 20 with a BCR-ABL1 transcript level > 10%. A KD mutation was found at 3 months in 4/41 patients (9.7%) all of whom progressed subsequently to advanced phase compared to only 3/37 in patients without mutation (p<0.001). Of note 3/4 patients with a KD mutation had a 3-months BCR-ABL1 transcript level >10%, while only one patient had a 3-months BCR-ABL transcript level <10%.In conclusion, ultra-deep NGS can reliably and consistently detect early appearance of KD mutation in patients who fail to achieve early molecular response at 3 months as well as in non responder patients or patients who are in CCyR but not in MMR, thus allowing potential early clinical intervention. DisclosuresBest:Onconova Therapeutics Inc: Research Funding. Pocock:Janssen: Honoraria. McLornan:Novartis: Research Funding, Speakers Bureau. Mufti:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. de Lavallade:Ariad: Research Funding; Novartis: Consultancy.

Molecular characterization of some fungi isolated from broiler chicken farms

The study was performed on 225 chicken (72 diseased and 153 freshly dead ones) from different 45commercial broiler farms (1- 30 days old) at Dakahlia Governorate for mycotic infection. Samples weretaken from these chickens (lung; air sac; crop; liver and brain from each chicken) after clinical andpostmortem examination for mycological examination. The results revealed that, fungi were isolatedfrom 503 positive samples (44.7%); represented as 119 positive samples (10.6%) from diseased chickensand 384 positive samples (34.1 %) from freshly dead ones.651 fungal isolates (moulds and yeasts) wereisolated from 1125 samples, where 219(33.6%) were isolated from lung samples followed by199(30.6%); 119(18.3%); 91(14.0%) and 23(3.5%) from air sac, liver, crop and brain samplesrespectively. Moreover, A. fumigatus was the most isolated one 141(21.7%) followed by A. flavus126(19.4%); A. niger 111(17.1%); Rhizopus spp. 67(10.3%); C. albicans 58(8.9%); Mucor spp.42(6.5%); Penicillium spp. 32(4.9%); A. terreus 20(3.1%); Fusarium spp.19 (2.9%); C. krusei 13(2.0%);C. tropicalis 9(1.4%); A. candidus 9(1.4%) and finally A. ochraceus 4(0.6%). PCR using fungus-specificuniversal primer pairs (ITS1 and ITS4) was used for identification and genotypic characters ofAspergillus species (A. fumigatus; A. flavus and A. niger).They were able to successfully amplify theITS1–5.8S rDNA region of all tested Aspergillus isolates, providing a single PCR product of about 600bp for all tested isolates PCR using fungus-specific universal primer pairs (ITS and RPS) was used foridentification and genotypic characters of C. providing a single PCR product of about 109 bp .Sequenceof the internal transcribed spacer 1 reigon (ITS-1) of isolated A. flavus Gene Bank accession numberfor studied nucleotide sequence (Bankit 1867303) is KM 983253. The sequences obtained for ITS-1region were more than 98% identical to the corresponding GenBank sequences (accession no.DQ467968.1; KC994648.1; LN482516.1 and KP689246.1). Sequence of the internal transcribed spacer1 region (ITS-1) of isolated C. albicans. Gene Bank accession number for studied nucleotide sequence(Bankit 1867955) is KM 983254. The sequences obtained for ITS-1 region obtained were more than95% identical to the corresponding GenBank (accession no. M87288.1; gb|S71769.1|S71769; emb(AL033396.1) and AP006852.1).

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes.

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

NCBI BLAST+ integrated into Galaxy.

BackgroundThe NCBI BLAST suite has become ubiquitous in modern molecular biology and is used for small tasks such as checking capillary sequencing results of single PCR products, genome annotation or even larger scale pan-genome analyses. For early adopters of the Galaxy web-based biomedical data analysis platform, integrating BLAST into Galaxy was a natural step for sequence comparison workflows.FindingsThe command line NCBI BLAST+ tool suite was wrapped for use within Galaxy. Appropriate datatypes were defined as needed. The integration of the BLAST+ tool suite into Galaxy has the goal of making common BLAST tasks easy and advanced tasks possible.ConclusionsThis project is an informal international collaborative effort, and is deployed and used on Galaxy servers worldwide. Several examples of applications are described here.

Rapid screening for protein solubility and expression

Abstract Abstract• References• Author information The Expresso® Solubility and Expression Screening System uses a simple ligase-free cloning strategy and a single PCR product to construct and test seven distinct protein fusion partners in parallel over the course of a few days. This multiplexed system simplifies the screening of fusion partners and greatly increases the chances of obtaining improved expression and solubility of target proteins. View full text At a glance Figures View all figures Figure 1: Features of the pSol expression vectors. Figure 2: Expresso solubility and expression workflow. The Expresso Solubility and Expression Screening System enables multiplexed evaluation of expression and solubility. Figure 3: Expression and purification of soluble GH1. (a) Screening for soluble GH1 fusion proteins. T, total protein; S, soluble fraction; I, insoluble pellet. Red stars indicate migration of GH1 fusion proteins. (b) Fusion-tag removal and purification of detagged GH1. Lanes 1 and 2 show purified Bla-GH1 fusion protein before and after cleavage with SelecTEV protease. Lanes 3, 4 and 5 show flow-through, wash and elution fractions from IMAC purification following the cleavage reaction. Free GH1 was present in the flow-through. M, marker proteins. (c) Purified, detagged GH1 remained soluble upon centrifugation after 72 h at 4 °C.

English

Arid soils are complex ecosystem that maintains topographically distinct mycoflora populations. A total of 45 soil samples collected from the arid soils in Iraqi desert were cultured by dilution plate method and screened for Aspergillus terreus. The aim of this study was to enhance ecological knowledge of cultured colonies variation of A. terreus as well as environmental interactions in arid soils. An attempt is made to comprehensively screen desert soil for the wild type A. terreus producing lovastatin. The results show that the most frequent Aspergillus spp. included: A. niger (159 isolates), A. terreus (143), A. flavus (115) and A. fumigates (42) and other fungi. Genetically, the diagnoses of 19 isolates of A. terreus were in the scope of our interest. The specific primer pair had monomorphic bands of approximately PCR product of 450 bp. Ribotyping isolates of A. terreus with primer pairs (ITS1/ITS4 and ITS1/ITS2) were 19 isolates, with a single PCR product of 550-600 bp and 280-380 bp, respectively. RAPD-PCR was also used to distinguish between ecological patterns generated and allowed a distinction of very closely related environmental isolates. Lovastatin production was carried out with thin layer chromatography. Results suggest that phenotypic variations in A. terreus isolates were not useful for identifying them, and showed no significance in the identification in all the sites studied. However, using discriminatory molecular methods, such as amplification of the targeted regions by specific and universal characterization of the isolates could be pivotal in understanding ecological variation. Wild type soil isolates of A. terreus have the ability to produce lovastatin. Key words: Molecular ecological typing, Aspergillus terreus, arid soils, lovastatin, Iraq.

A novel localization of molt-inhibiting hormone in the tegumental glands of shrimp Penaeus monodon and its possible role in shrimp molting

Abstract Molt-inhibiting hormone (MIH) is commonly localized in the X-organ sinus gland complex (XOSG) of the crustacean. The present study aimed to elucidate the expression of MIH at the subcellular and cellular levels in the eyestalks and integument of juvenile Penaeus monodon during the molting cycle. Gene expression of Pem-MIH1 in the optic lobes showed a single PCR product at 172 bp, and was restricted only to the eyestalk XOSG but not detected in pleopods, cephalothorax integument, muscle, hepatopancreas or retina. Quantitative analysis of gene expression in the eyestalks demonstrated no significant alteration of Pem-MIH1 mRNA in XOSG during the molt cycle. Immunohistochemistry using antibody against recombinant molt-inhibiting hormone-like peptide (anti-MIH-like) revealed variable staining intensities of individual MTXOs but were most persistently intense in SGs. During the molt cycle, the mean numbers of XO immunoreactive cells were slightly oscillated but not statistically different. The quantitative immunohistochemistry measured from XOSG illustrated minimal fluctuations of the values obtained suggesting the periodical synthesis and release of MIH from XOSG system during the molt cycle. The positive reaction was also detected in the tegumental glands located at the retina and the integument of walking leg, base of eyestalk and cephalothorax of the shrimp. The large variations of immunostaining and amount of reactive tegumental glands in the eyestalk were noted throughout the molt cycle. The numbers of the MIH-like immunoreactive glands obtained from the retina as well as the integument base did not significantly change during the molt cycle but tend to increase during postmolt to intermolt and decrease during premolt. These results suggest that the MIH-like peptides in the tegumental glands are probably released mostly during the late premolt. Our findings thus propose the novel storage site of MIH besides SG of the eyestalk including a new target tissue, an epidermal cell, the role of which may link to the complicated molting regulation and/or the dark-adaptation of shrimp.