ResearchPad - dna-isolation https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Rediscovering an old foe: Optimised molecular methods for DNA extraction and sequencing applications for fungarium specimens of powdery mildew (Erysiphales)]]> https://www.researchpad.co/article/elastic_article_14476 The purpose of this study was to identify a reliable DNA extraction protocol to use on 25-year-old powdery mildew specimens from the reference collection VPRI in order to produce high quality sequences suitable to address taxonomic phylogenetic questions. We tested 13 extraction protocols and two library preparation kits and found the combination of the E.Z.N.A.® Forensic DNA kit for DNA extraction and the NuGen Ovation® Ultralow System library preparation kit was the most suitable for this purpose.

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<![CDATA[Genome-enhanced detection and identification of fungal pathogens responsible for pine and poplar rust diseases]]> https://www.researchpad.co/article/5c648cebd5eed0c484c81ab7

Biosurveillance is a proactive approach that may help to limit the spread of invasive fungal pathogens of trees, such as rust fungi which have caused some of the world’s most damaging diseases of pines and poplars. Most of these fungi have a complex life cycle, with up to five spore stages, which is completed on two different hosts. They have a biotrophic lifestyle and may be propagated by asymptomatic plant material, complicating their detection and identification. A bioinformatics approach, based on whole genome comparison, was used to identify genome regions that are unique to the white pine blister rust fungus, Cronartium ribicola, the poplar leaf rust fungi Melampsora medusae and Melampsora larici-populina or to members of either the Cronartium and Melampsora genera. Species- and genus-specific real-time PCR assays, targeting these unique regions, were designed with the aim of detecting each of these five taxonomic groups. In total, twelve assays were developed and tested over a wide range of samples, including different spore types, different infected plant parts on the pycnio-aecial or uredinio-telial host, and captured insect vectors. One hundred percent detection accuracy was achieved for the three targeted species and two genera with either a single assay or a combination of two assays. This proof of concept experiment on pine and poplar leaf rust fungi demonstrates that the genome-enhanced detection and identification approach can be translated into effective real-time PCR assays to monitor tree fungal pathogens.

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<![CDATA[PepN is a non-essential, cell wall-localized protein that contributes to neutrophil elastase-mediated killing of Streptococcus pneumoniae]]> https://www.researchpad.co/article/5c5df336d5eed0c484580f0d

Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause disease in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance of Spn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis, Spn is exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killing Spn, the bacterial proteins that are degraded by NSPs leading to Spn death are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN a CW-localized protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotype in vitro in the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophils ex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing of Spn by NSPs and human neutrophils.

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<![CDATA[Developing tools for evaluating inoculation methods of biocontrol Streptomyces sp. strains into grapevine plants]]> https://www.researchpad.co/article/5c536bfcd5eed0c484a496c7

The endophytic Streptomyces sp. VV/E1, and rhizosphere Streptomyces sp. VV/R4 strains, isolated from grapevine plants were shown in a previous work to reduce the infection rate of fungal pathogens involved in young grapevine decline. In this study we cloned fragments from randomly amplified polymorphic DNA (RAPD), and developed two stably diagnostic sequence-characterized amplified region (SCAR) markers of 182 and 160 bp for the VV/E1 and VV/R4 strains, respectively. The SCAR markers were not found in another 50 actinobacterial strains isolated from grapevine plants. Quantitative real-time PCR protocols based on the amplification of these SCAR markers were used for the detection and quantification of both strains in plant material. These strains were applied on young potted plants using two methods: perforation of the rootstock followed by injection of the microorganisms or soaking the root system in a bacterial suspension. Both methods were combined with a booster treatment by direct addition of a bacterial suspension to the soil near the root system. Analysis of uprooted plants showed that those inoculated by injection exhibited the highest rate of colonization. In contrast, direct addition of either strain to the soil did not lead to reliable colonization. This study has developed molecular tools for analyzing different methods for inoculating grapevine plants with selected Streptomyces sp. strains which protect them from fungal infections that enter through their root system. These tools are of great applied interest since they could easily be established in nurseries to produce grafted grapevine plants that are protected against fungal pathogens. Finally, this methodology might also be applied to other vascular plants for their colonization with beneficial biological control agents.

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<![CDATA[Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL)]]> https://www.researchpad.co/article/5bf71f9ad5eed0c484dcb98e

Background

The World Health Organization has targeted elimination of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) by 2020. Despite distinctive decline seen in the number of VL cases in ISC, there is still a quest for development of a diagnostic test which has the utility for detection of active infection and relapse cases and as a test of cure. The present study validated the sensitivity and specificity of SYBR Green I based closed tube LAMP assay reported by us for diagnosis of VL.

Methodology

The validation study was carried out at two endemic sites in India, located at Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna and Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi. Standard operating protocols were provided at the two sites for applying LAMP assay on confirmed VL cases. The diagnostic accuracy of LAMP assay was evaluated by Receiver operator curve (ROC) analysis. Furthermore, a simplified LAMP assay based on direct blood lysis, DBL-LAMP, was developed and verified for its diagnostic accuracy.

Principal findings

A total of 267 eligible participants were included in the study which comprised of 179 VL cases and 88 controls. Sensitivity and specificity of the LAMP assay were 98.32% (95% C.I– 95.2–99.7%) and 96.59% (95% C.I.-90.4–99.3%), respectively. ROC curve analysis depicted no significant difference between area under curve (AUCROC) for LAMP assay and rK39 RDT, indicative of LAMP as an excellent diagnostic test. DBL-LAMP assay, performed on 67 VL and 100 control samples, yielded a sensitivity of 93.05% (95% C.I- 84.75–97%) and specificity of 100% (95% C.I.- 96.30–100%).

Conclusions/Significance

The validated closed tube LAMP for diagnosis of VL will provide impetus to the ongoing VL elimination programme in ISC. The assay based on direct blood lysis promotes its scope for application in field settings by further reducing time and cost.

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<![CDATA[Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples]]> https://www.researchpad.co/article/5989da75ab0ee8fa60b963a7

Background

Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.

Methods

Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.

Results

An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens.

Conclusions

The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.

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<![CDATA[Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria]]> https://www.researchpad.co/article/5989daa8ab0ee8fa60ba83d9

Background

Red blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria.

Results

Flow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents.

Conclusions

These studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators.

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<![CDATA[Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles]]> https://www.researchpad.co/article/5989da42ab0ee8fa60b8a480

The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.

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<![CDATA[Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase]]> https://www.researchpad.co/article/5989da7dab0ee8fa60b994c2

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification.

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<![CDATA[Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material]]> https://www.researchpad.co/article/5989da88ab0ee8fa60b9cff8

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

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<![CDATA[Human Treponema pallidum 11q/j isolate belongs to subsp. endemicum but contains two loci with a sequence in TP0548 and TP0488 similar to subsp. pertenue and subsp. pallidum, respectively]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcdba

Background

Treponema pallidum subsp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). An unusual human TEN 11q/j isolate was obtained from a syphilis-like primary genital lesion from a patient that returned to France from Pakistan.

Methodology/Principal findings

The TEN 11q/j isolate was characterized using nested PCR followed by Sanger sequencing and/or direct Illumina sequencing. Altogether, 44 chromosomal regions were analyzed. Overall, the 11q/j isolate clustered with TEN strains Bosnia A and Iraq B as expected from previous TEN classification of the 11q/j isolate. However, the 11q/j sequence in a 505 bp-long region at the TP0488 locus was similar to Treponema pallidum subsp. pallidum (TPA) strains, but not to TEN Bosnia A and Iraq B sequences, suggesting a recombination event at this locus. Similarly, the 11q/j sequence in a 613 bp-long region at the TP0548 locus was similar to Treponema pallidum subsp. pertenue (TPE) strains, but not to TEN sequences.

Conclusions/Significance

A detailed analysis of two recombinant loci found in the 11q/j clinical isolate revealed that the recombination event occurred just once, in the TP0488, with the donor sequence originating from a TPA strain. Since TEN Bosnia A and Iraq B were found to contain TPA-like sequences at the TP0548 locus, the recombination at TP0548 took place in a treponeme that was an ancestor to both TEN Bosnia A and Iraq B. The sequence of 11q/j isolate in TP0548 represents an ancestral TEN sequence that is similar to yaws-causing treponemes. In addition to the importance of the 11q/j isolate for reconstruction of the TEN phylogeny, this case emphasizes the possible role of TEN strains in development of syphilis-like lesions.

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<![CDATA[Implementation of Novel Design Features for qPCR-Based eDNA Assessment]]> https://www.researchpad.co/article/5989d9e1ab0ee8fa60b69ce5

Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated on the specificity and sensitivity of the PCR assay, careful validation of each eDNA qPCR assay in development must be performed both under controlled laboratory conditions and when challenged with field-derived eDNA samples. Such a step-wise approach forms the basis for incorporation of innovative qPCR design features that strengthen the implementation and interpretation of the eDNA assay. This includes empirical determination that the qPCR assay is refractory to the presence of human DNA and the use of a tripartite assay approach comprised of 1) a primer set targeting plant chloroplast that evaluates the presence of amplifiable DNA from field samples to increase confidence in a negative result, 2) an animal group primer set to increase confidence in the assay result, and 3) a species-specific primer set to assess presence of DNA from the target species. To demonstrate this methodology, we generated eDNA assays specific for the North American bullfrog (Lithobates (Rana) catesbeiana) and the Rocky Mountain tailed frog (Ascaphus montanus) and characterized each with respect to detection sensitivity and specificity with demonstrated performance in a field survey scenario. The qPCR design features presented herein address specific challenges of eDNA assays thereby increasing their interpretative power.

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<![CDATA[The Emergence and Spread of Multiple Livestock-Associated Clonal Complex 398 Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains among Animals and Humans in the Republic of Ireland, 2010–2014]]> https://www.researchpad.co/article/5989dacfab0ee8fa60bb5906

Clonal complex (CC) 398 methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) are associated with carriage and infection among animals and humans but only a single case of CC398 MRSA has been reported in the Republic of Ireland (ROI). The present study investigated the molecular epidemiology of CC398 MRSA (n = 22) and MSSA (n = 10) from animals and humans in the ROI from 2010–2014. Isolates underwent antimicrobial susceptibility testing, spa typing, DNA microarray profiling and PCR for CC398-associated resistance genes. All MRSA underwent SCCmec IV or V subtyping. Four distinct CC398-MRSA incidents were identified from (i) a man in a nursing home (spa type t011-SCCmec IVa, immune evasion complex (IEC) negative), (ii) a horse and veterinarian who had recently travelled to Belgium (t011-IVa, IEC positive), (iii) pigs (n = 9) and farm workers (n = 9) on two farms, one which had been restocked with German gilts and the other which was a finisher farm (t034-VT, IEC negative, 3/9 pigs; t011- VT, IEC negative, 6/9 pigs & 9/9 farm workers), and (iv) a child who had worked on a pig farm in the UK (t034-VT, IEC negative). Isolates also carried different combinations of multiple resistance genes including erm(A), erm(B), tet(K), tet(M) & tet(L), fexA, spc, dfrG, dfrK aacA-aphD and aadD further highlighting the presence of multiple CC398-MRSA strains. CC398 MSSA were recovered from pigs (n = 8) and humans (n = 2). CC398 MSSA transmission was identified among pigs but zoonotic transmission was not detected with animal and human isolates exhibiting clade-specific traits. This study highlights the importation and zoonotic spread of CC398 MRSA in the ROI and the spread of CC398 MSSA among pigs. Increased surveillance is warranted to prevent further CC398 MRSA importation and spread in a country that was considered CC398 MRSA free.

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<![CDATA[Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonella koehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats]]> https://www.researchpad.co/article/5989dae0ab0ee8fa60bbbbd6

Domestic cats are the natural reservoir of Bartonella henselae, B. clarridgeiae and B. koehlerae. To determine the role of wild felids in the epidemiology of Bartonella infections, blood was collected from 14 free-ranging California mountain lions (Puma concolor) and 19 bobcats (Lynx rufus). Bartonella spp. were isolated from four (29%) mountain lions and seven (37%) bobcats. These isolates were characterized using growth characteristics, biochemical reactions, molecular techniques, including PCR-RFLP of selected genes or interspacer region, pulsed-field gel electrophoresis (PFGE), partial sequencing of several genes, and DNA-DNA hybridization. Two isolates were identical to B. henselae genotype II. All other isolates were distinguished from B. henselae and B. koehlerae by PCR-RFLP of the gltA gene using endonucleases HhaI, TaqI and AciI, with the latter two discriminating between the mountain lion and the bobcat isolates. These two novel isolates displayed specific PFGE profiles distinct from B. henselae, B. koehlerae and B. clarridgeiae. Sequences of amplified gene fragments from the three mountain lion and six bobcat isolates were closely related to, but distinct from, B. henselae and B. koehlerae. Finally, DNA-DNA hybridization studies demonstrated that the mountain lion and bobcat strains are most closely related to B. koehlerae. We propose naming the mountain lion isolates B. koehlerae subsp. boulouisii subsp. nov. (type strain: L-42-94), and the bobcat isolates B. koehlerae subsp. bothieri subsp. nov. (type strain: L-17-96), and to emend B. koehlerae as B. koehlerae subsp. koehlerae. The mode of transmission and the zoonotic potential of these new Bartonella subspecies remain to be determined.

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<![CDATA[DNA methylation levels in candidate genes associated with chronological age in mammals are not conserved in a long-lived seabird]]> https://www.researchpad.co/article/5ab4d55b463d7e0969868611

Most seabirds do not have any outward identifiers of their chronological age, so estimation of seabird population age structure generally requires expensive, long-term banding studies. We investigated the potential to use a molecular age biomarker to estimate age in short-tailed shearwaters (Ardenna tenuirostris). We quantified DNA methylation in several A. tenuirostris genes that have shown age-related methylation changes in mammals. In birds ranging from chicks to 21 years of age, bisulphite treated blood and feather DNA was sequenced and methylation levels analysed in 67 CpG sites in 13 target gene regions. From blood samples, five of the top relationships with age were identified in KCNC3 loci (CpG66: R2 = 0.325, p = 0.019). In feather samples ELOVL2 (CpG42: R2 = 0.285, p = 0.00048) and EDARADD (CpG46: R2 = 0.168, p = 0.0067) were also weakly correlated with age. However, the majority of markers had no clear association with age (of 131 comparisons only 12 had a p-value < 0.05) and statistical analysis using a penalised lasso approach did not produce an accurate ageing model. Our data indicate that some age-related signatures identified in orthologous mammalian genes are not conserved in the long-lived short tailed shearwater. Alternative molecular approaches will be required to identify a reliable biomarker of chronological age in these seabirds.

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<![CDATA[An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples]]> https://www.researchpad.co/article/5989da66ab0ee8fa60b91f38

Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from <1 cell /ml for river water to 36 cells/mL for ultrapure water with E. coli as a carrier. In conclusion, we optimized a method to quantify pathogenic Leptospira in environmental waters (river, pond and sewage) which consists of the concentration of 40 mL samples by centrifugation at 15,000×g for 20 minutes at 4°C, followed by DNA extraction with the PowerSoil DNA Isolation kit. Although the method described herein needs to be validated in environmental studies, it potentially provides the opportunity for effective, timely and sensitive assessment of environmental leptospiral burden.

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<![CDATA[Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer]]> https://www.researchpad.co/article/5989db1aab0ee8fa60bce005

We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample.

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<![CDATA[Molecular mechanisms of cooperative binding of transcription factors Runx1–CBFβ–Ets1 on the TCRα gene enhancer]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbb31

Ets1 is an essential transcription factor (TF) for several important physiological processes, including cell proliferation and differentiation. Its recognition of the enhancer region of the TCRα gene is enhanced by the cooperative binding of the Runx1–CBFβ heterodimer, with the cancelation of phosphorylation-dependent autoinhibition. The detailed mechanism of this interesting cooperativity between Ets1 and the Runx1–CBFβ heterodimer is still largely unclear. Here, we investigated the molecular mechanisms of this cooperativity, by using molecular dynamics simulations. Consequently, we detected high flexibility of the loop region between the HI2 and H1 helices of Ets1. Upon Runx1–CBFβ heterodimer binding, this loop transiently adopts various sub-stable conformations in its interactions with the DNA. In addition, a network analysis suggested an allosteric pathway in the molecular assembly and identified some key residues that coincide with previous experimental studies. Our simulations suggest that the cooperative binding of Ets1 and the Runx1–CBFβ heterodimer alters the DNA conformation and induces sub-stable conformations of the HI2–H1 loop of Ets1. This phenomenon increases the flexibility of the regulatory module, including the HI2 helix, and destabilizes the inhibitory form of this module. Thus, we hypothesize that this effect facilitates Ets1–DNA binding and prevents the phosphorylation-dependent DNA binding autoinhibition.

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<![CDATA[Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing]]> https://www.researchpad.co/article/5989db00ab0ee8fa60bc62c3

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.

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<![CDATA[Genetic Variation within Clonal Lineages of Phytophthora infestans Revealed through Genotyping-By-Sequencing, and Implications for Late Blight Epidemiology]]> https://www.researchpad.co/article/5989da73ab0ee8fa60b95b40

Genotyping-by-sequencing (GBS) was performed on 257 Phytophthora infestans isolates belonging to four clonal lineages to study within-lineage diversity. The four lineages used in the study were US-8 (n = 28), US-11 (n = 27), US-23 (n = 166), and US-24 (n = 36), with isolates originating from 23 of the United States and Ontario, Canada. The majority of isolates were collected between 2010 and 2014 (94%), with the remaining isolates collected from 1994 to 2009, and 2015. Between 3,774 and 5,070 single-nucleotide polymorphisms (SNPs) were identified within each lineage and were used to investigate relationships among individuals. K-means hierarchical clustering revealed three clusters within lineage US-23, with US-23 isolates clustering more by collection year than by geographic origin. K-means hierarchical clustering did not reveal significant clustering within the smaller US-8, US-11, and US-24 data sets. Neighbor-joining (NJ) trees were also constructed for each lineage. All four NJ trees revealed evidence for pathogen dispersal and overwintering within regions, as well as long-distance pathogen transport across regions. In the US-23 NJ tree, grouping by year was more prominent than grouping by region, which indicates the importance of long-distance pathogen transport as a source of initial late blight inoculum. Our results support previous studies that found significant genetic diversity within clonal lineages of P. infestans and show that GBS offers sufficiently high resolution to detect sub-structuring within clonal populations.

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