Low DNA Concentrations Research Articles

Identifying individual ungulates from fecal DNA: a comparison of field collection methods to maximize efficiency, ease, and success

Non-invasive genetic sampling can facilitate the identification of individual animals across a landscape, with applications to management and conservation. Fecal material is a readily available source of DNA, and various methods exist for collecting fecal samples for DNA preservation. In particular, swab methods offer considerable promise, but their utility in real-world field contexts remains relatively untested. We systematically compared multiple genetic fecal sampling methods across all stages of data collection and analysis, including sampling in the field, DNA extraction in the lab, and identification of individuals using microsatellite genotyping. We collected 112 fecal samples from black-tailed deer (Odocoileus hemionus columbianus) in the field in Mendocino County, California, across a range of sample conditions of unknown age. We systematically compared the efficiency, ease, and genotyping success of three methods for field collection and storage of ungulate fecal samples: whole pellets in ethanol, whole dry pellets in paper envelopes, and cotton swabs in buffer. Storage method, sample condition, and their interaction predicted genotyping success in the top binomial GLMMs. We found that swabbing pellets resulted in the greatest percentage of individually identifiable genotypes (81%, compared to 60% for dry samples and 56% for ethanol), despite lower DNA concentrations. While swabbing pellets requires a greater time investment in the field, the samples are easier and safer to store and transport, and subsequent labwork is more efficient as compared to whole-pellet collection methods. We, therefore, recommend the swab method for most contexts. We provide additional recommendations and field protocols based on subsequent collection of 2284 swab samples for a larger monitoring study of the deer population, given that this large number of samples spanned a range of sample conditions and time spent in storage.

Self-Assembled Single-Stranded DNA Nano-Networks in Solution and at Surfaces.

We studied the directed self-assembly of two types of complementary single-stranded DNA (ssDNA) strands [i.e., poly(dA) and poly(dT)] into more complex, organized, and percolating networks in dilute solutions and at surfaces. Understanding ssDNA self-assembly into 2D networks on surfaces is important for the use of such networks in the fabrication of well-defined nanotechnological devices, as, for instance, required in nanoelectronics or for biosensing. To control the formation of 2D networks on surfaces, it is important to know whether DNA assemblies are formed already in dilute solutions or only during the drying/immobilization process at the surface, where the concentration automatically increases. Fluorescence cross-correlation spectroscopy clearly shows the presence of larger DNA complexes in mixed poly(dA) and poly(dT) solutions already at very low DNA concentrations (<1 nM), that is, well below the overlap concentration. Here, we describe for the first time such supramolecular complexes in solution and how their structure depends on the ssDNA length and concentration and ionic strength. Hence, future attempts to control such networks should also focus on network precursors in solution and not only on their immobilization on surfaces.

Evaluation of the TruSight Tumor 170 Assay and Its Value in Clinical Diagnostics

Background: Parallel sequencing technologies have become integrated into clinical practice. This study evaluated the TruSight Tumor 170 assay for the simultaneous detection of somatic gene mutations (SNPs and indels), gene fusions and CNVs, and its implementation into routine diagnostics. Methods: Forty-four formalin-fixed, paraffin-embedded tissue samples analyzed previously with validated methods were evaluated with the TruSight Tumor 170 assay (Illumina). For data analysis the TruSight Tumor 170 app, the BaseSpace Variant Interpreter (Illumina), and the Molecular Health Guide Software (Molecular Health) were used. Results: All somatic gene mutations were identified when covered by the assay. Two high-level MET amplifications were detected by CNV analysis. Focal MET amplifications with a copy number below 10 were not reliably detected at the DNA-level. Twenty-one of 31 fusions and splice variants were confirmed with the assay on the RNA-level. The remaining eight aberrations were incorrect by previous methods. In two cases, no splicing was observed. Conclusions: The TruSight Tumor 170 gives reliable results even if low DNA and RNA concentrations are applied in comparison to other methods and can be used in a routine workflow to detect somatic gene mutations, gene fusions, and splice variants. However, we were not able to detect most focal gene amplifications/deletions.

Modeling multiple duplex DNA attachments in a force-extension experiment

Optical tweezers-based DNA stretching often relies on tethering a single end-activated DNA molecule between optically manipulated end-binding beads. Measurement success can depend on DNA concentration. At lower DNA concentrations tethering is less common, and many trials may be required to observe a single-molecule stretch. At higher DNA concentrations tethering is more common; however, the resulting force-extensions observed are more complex and may vary from measurement to measurement. Typically these more complex results are attributed to the formation of multiple tethers between the beads; however, to date there does not appear to have been a critical examination of this hypothesis or the potential usefulness of such data. Here we examine stretches at a higher DNA concentration and use analysis and simulation to show how the more complex force-extensions observed can be understood in terms of multiple DNA attachments.

Zinc oxide and gold textured Janus nanowire integration in an impedimetric sensor for leptospirosis DNA-biomarker recognition.

A "Janus particle" refers to the production of two materials in a single system and shows a difference in physical characteristics, and two surfaces participate in the formation with different chemistries. This research generated the Janus using a hybrid of zinc oxide (ZnO) and gold (Au) on the sensor surface toward making high-performance DNA sensors. The Janus ZnO/Au-textured film was synthesized via the one-step sol-gel method, which involves a suitable ratio of a mixture of ZnO sol seed solution. The synthesized Janus ZnO/Au-textured film undergoes a low-temperature aqueous hydrothermal route to synthesize quasi-one-dimensional nanowires. The average grain size in the Janus ZnO/Au nanotextured wire was 41.60 nm. The fabricated nanotextured wire was further optimized by tuning the thickness and characterized by XRD and high-resolution microscopy. Electrical characterization was conducted on the Janus ZnO/Au nanotextured wire coupled with an interdigitated electrode sensor to detect the specific leptospirosis DNA strand. The generated device is capable of detecting lower DNA concentration at 1 × 10-13 M with a sensitivity of 8.54 MΩ M-1 cm-2 . The high performance is attained on linear concentrations of 10-6 -10-13 M with the determination coefficient, "I = 135437.63C-3609.07" R2 = 0.9551. A potential strategy is proposed as a base for developing different high-performance sensors.

Comparative Analysis of a Developed Probe-Based Real-Time PCR and PCR-SSP for HLA-B*27 Detection among Thai Blood Donors

The human leukocyte antigen (HLA) class I gene, the B locus, allele 27, HLA-B*27 is one of the most fascinating risk factors that is strongly associated with developing spondyloarthropathies (SpA). HLA-B27 testing has been routinely available in the diagnosis of those diseases. This study aimed to develop a fluorogenic real-time PCR and to compare it with PCR-SSP to detect the HLA-B*27 allele among Thai blood donors. A total of 391 DNA samples were obtained from Thai blood donors at Thammasat University Hospital and tested for HLA-B*27 allele detection. A new real-time PCR was developed and validated to identify this allele and subsequently compared with those results tested with PCR-SSP. The sensitivity of detection was performed using known HLA-B*27-positive and -negative samples with concentrations ranging from 0.001 to 100 ng/µL. Additionally, HLA-B27 subtyping was performed by DNA sequencing containing second and third exons of this gene among all the HLA-B*27-positive donors. The validity of real-time PCR using known DNA controls and the results obtained by PCR-SSP techniques were in 100% concordance. The method was sensitive even at low DNA concentrations (1 ng/µL). Of 391 donors, 24 (6.14%; 95% CI, 3.97 - 9.00) were found to have the HLA-B*27 allele, while the remaining 367 (93.86%; 95% CI, 91.00 - 96.03) did not have this allele. Donors presented HLA-B*27-positive, HLA-B*27:06, the most common allele, followed by HLA-B*27:04, -B*27:05, and -B*27:07. HLA-B*27 using fluorogenic real-time qualitative PCR was found to be superior compared with that of PCR-SSP. The method is rapid, accurate, reliable, and sensitive for detection. In addition, this method provides convenience in the early treatment of SpA patients and relieves their suffering.

Development and application of TaqMan probe-based quantitative PCR assays for the detection of tilapia parvovirus.

Tilapia parvovirus (TiPV) is a novel parvovirus associated with high mortality in Nile tilapia and red hybrid tilapia, leading to severe economic losses for tilapia aquaculture. It is critical to develop a sensitive and accurate assay to detect TiPV in fish tissues. In this study, new TaqMan probe-based quantitative PCR (qPCR) assays targeting the non-structural (NS) and viral protein (VP) genes of TiPV were developed. The standard curves of the assays were 95.64%-98.96% over a wide linear range of 109 -101 copies of the corresponding standard DNA per reaction. The intra- and inter-assay coefficients of variation were in the ranges 0.54%-2.50% and 0.13%-1.17%, respectively, which suggests good repeatability and reproducibility. The detection limit of the TaqMan TiPV assays was 10 copies/µl. The application of the TaqMan qPCR assays to field samples revealed that they had comparable sensitivity to a previously developed SYBR Green qPCR, but more sensitive than the conventional PCR. No cross-reactivity of the TaqMan TiPV assays was found with the samples infected with other viruses and bacteria. Overall, the assays offered high sensitivity and specificity in the detection of low concentrations of TiPV DNA in infected tilapia samples. These new TaqMan qPCR assays could provide a valuable diagnostic tool for the reliable and specific detection of TiPV in experimental and field samples.

Deceptive combined effects of short allele dominance and stuttering: an example with Ixodes scapularis, the main vector of Lyme disease in the U.S.A.

Null alleles, short allele dominance (SAD), and stuttering increase the perceived relative inbreeding of individuals and subpopulations as measured by Wright9s FIS and FST. Ascertainment bias, due to such amplifying problems are usually caused by inaccurate primer design (if developed from a different species or a distant population), poor DNA quality, low DNA concentration, or a combination of some or all these sources of inaccuracy. When combined, these issues can increase the correlation between polymorphism at concerned loci and, consequently, of linkage disequilibrium (LD) between those. In this note, we studied an original microsatellite data set generated by analyzing nine loci in Ixodes scapularis ticks from the eastern U.S.A. To detect null alleles and SAD we used correlation methods and variation measures. To detect stuttering, we evaluated heterozygote deficit between alleles displaying a single repeat difference. We demonstrated that an important proportion of loci affected by amplification problems (one with null alleles, two with SAD and three with stuttering) lead to highly significant heterozygote deficits (FIS=0.1, p-value

Herpesviruses in patients after renal transplantation

To estimate graft function after kidney transplantation during active herpesviruses or superinfection Materials and methods. The study included 32 patients (men 21, women 11) with end-stage chronic kidney disease. The median age was 43 years. Cytomegalovirus (CMV), EpsteinBarr virus (EBV) and human herpes virus 6 (HHV-6) DNAs were screened by RT-PCR in the donor's transplant biopsy, and recipients peripheral blood and urine after kidney transplantation (KT) on 0, 1, 2, 4, 6, 12 months. Antiviral antibodies (IgM and IgG) were also screened by Enzyme-linked immunoassay analysis (ELISA) along with PCR. The 500 or less copies of viral DNA per 105 nuclear cells or 1 ml of urine was considered as low, more than 1000 copies high. On the first month after KT CMV DNA was detected in 50% of pts., EBV DNA in 40% and HHV-6 DNA in 33%. During first year after KT two or three viruses simultaneously were found in 12 recipients: CMV, EBV, and HHV-6 were detected in 5 recipients; CMV and EBV in 4 patients; CMV and HHV-6 in 2 pts; EBV and HHV-6 in 1 pt. Graft dysfunction was observed in 9 patients with a high concentration of viral DNA of one, two or three viruses simultaneously. An upraise of the concentration of virus DNA (CMV, EBV and HHV 6) was detected primarily in the urine, while in the blood its concentration was less than 500 cop or undetectable. Renal dysfunction was not observed on the background of low concentrations of viral DNA in urine and blood. However, with an increase of DNA concentration, an impaired graft function in 8 of 12 patients appeared. Low viral DNA level proved to be a background for another virus activation or bacterial/fungal superinfection. Graft dysfunction occurs at high viral DNA levels detection during mono-or superinfection. Low viral load can serve as a background for another virus activation and/or bacterial/fungal superinfection.

Multiplex PCR development for the simultaneous and rapid detection of two pathogenic flukes, Dactylogyrus spp. and Centrocestus formosanus, in ornamental fishes

Both monogenean Dactylogyrus spp. and digenean Centrocestus formosanus are major pathogens for aquaculture and the fish industry, especially in goldfish (Carassius auratus). The resulting infections in the fish cause extensive morbidity and mortality. However, the accurate detection of both parasitic diseases requires highly experienced personnel and takes a long time. Reliable and sensitive diagnostic methods are needed for detecting the infections in various fish species. To overcome this problem, this study developed the multiplex polymerase chain reaction (m-PCR) assay to rapidly identify and diagnose Dactylogyrus spp. and C. formosanus simultaneously on the target genes (18S and cytb gene) in field-collected samples under one reaction. Two pairs of species-specific primers were manually designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results demonstrated that the multiplex PCR assay can be used to detect Dactylogyrus spp. and C. formosanus specifically, without cross-amplification with other parasite-related species and their host. The sensitivity showed that the m-PCR system could amplify the two target genes with the lowest DNA concentration at 0.078 ng/μL for Dactylogyrus spp. and 0.625 ng/μL for C. formosanus. The detection limit in the case of genomic combination between samples with goldfish was detected at the ratios of 1:13 and 1:2 ng/μL for Dactylogyrus spp. and C. formosanus, respectively. Moreover, this assay can also be developed and applied to detect the adult, metacercariae, cercariae, and egg stages of C. formosanus as well. Additionally, a total of 50 goldfish samples collected from several farms were found to be positive for Dactylogyrus spp., C. formosanus, and co-infection (fifteen, fourteen, and eighteen, respectively). The developed multiplex PCR shows advantages in specificity and sensitivity which prove to be an invaluable tool for diagnoses and monitoring the epidemic situation.

Influence of deep-freezing and MGG staining on DNA and RNA quality in different types of lung adenocarcinoma cytological smears.

Preserving the optimal quality of DNA and RNA is mandatory for molecular testing in lung adenocarcinoma cytological smears (LACSs). DNA and RNA were isolated from 90 frozen unstained and 46 May Grünwald Giemsa (MGG) stained LACSs prepared from bronchial washing (BW), bronchial brushing (BB), and pleural effusion (PE) samples during 3 years. Concentrations of nucleic acids in all LACSs were assessed by spectrophotometric analysis. Fragmentation of DNA and RNA was determined by PCR amplification of selected genes. Amplicons of 100, 200, 300, 400, and 600 bp were used for DNA and 108 bp-long HPRT1 transcript fragment for RNA fragmentation analysis. Among 90 frozen LACSs, significantly lower DNA concentrations of BB and RNA concentrations of BW samples frozen for 6-10 months were observed in comparison with samples frozen for longer periods (p< .05). Among 46 paired LACSs, 44 (95.7%) frozen and 15 (32.6%) MGG-stained samples showed 600 bp-long DNA amplicons. Statistically significant difference (p< .05) in the fragmentation of DNA between frozen and MGG-stained LACSs was observed (p< .05), with DNA being less fragmented in frozen LACSs. In addition, 33 (71.7%) frozen and 36 (78.2%) MGG-stained LASCs showed HPRT1 gene amplicon of 108 bp. RNA was less fragmented in 3-year old MGG-stained samples than in LACSs frozen for 3 years. DNA and RNA extracted from frozen and MGG-stained LACSs showed different results depending on the time of storage and/or type of samples, but in general all samples had adequate quantity and quality for downstream molecular testing.

Circulating Tumour DNA for Early Diagnosis of Pancreatic Ductal Adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDAC) has been deathly cancer arising from pancreatic cells. Despite the improvement in the standard of diagnosis, most patients seek medical care in the late stage. Due to the aggressiveness of the disease, it is therefore imperative to detect the early lesion for a better outcome.Methods: We identified 416 articles relevant to circulating tumor DNA (ctDNA) and PDAC using predefined keywords in PubMed, PubMed Central, and Cochrane Library from January 1, 2011, to January 1, 2021 (10 years). Firstly, we screened the titles and abstracts, and 63 articles were included. Then, we screened those articles for the full-text version and included only 8 articles fulfilling our inclusion criteria. All steps were reviewed by the author.Results: The presence of ctDNA in the blood reflects the occurrence of the pancreatic cancerspecific mutation in the primary tumor. The detection of KRAS mutation in ctDNA and tumor samples is highly consistent. The number of positive findings in early-stage patients is low, in line with the low ctDNA concentration measured. However, the combination of KRAS detected in ctDNA and other biomarkers showed prominent results with higher sensitivity and specificity.Conclusions: ctDNA is a promising tool for early detection of PDAC. Despite its low positivity rate in certain studies, it is considerably concordant with the primary tumor. Future improvement in the technique application is required to overcome the issue of low DNA concentration in circulation.

Evaluation of Droplet Digital PCR Assay for the Diagnosis of Candidemia in Blood Samples.

Numerous studies have shown that droplet digital PCR (ddPCR) is a promising tool for the diagnosis of pathogens, especially in samples with low concentrations of pathogenic DNA. An early diagnosis of candidemia is critical for the effective treatment of patients. In this study, we evaluated the sensitivity and specificity of ddPCR assay for Candida DNA detection both in vitro by mixing fungal cells with human blood and in vivo by analyzing blood samples from infected mice and patients with suspected candidemia. The results showed that ddPCR assay could detect a minimum of 4.5 DNA copies per reaction in blood samples. ddPCR showed higher sensitivity and specificity for Candida DNA detection than traditional culture and quantitative PCR (qPCR) methods and also exhibited significantly better positive and negative predictive values than the culture and qPCR methods that were commonly used in clinical practice. Hence, our study demonstrates that ddPCR assay is a promising method for the timely diagnosis of candidemia and could be useful for monitoring the treatment of candidemia.

PD43-04 A URINE-BASED DNA METHYLATION MARKER TEST TO DETECT UPPER TRACT UROTHELIAL CARCINOMA: A PILOT STUDY

You have accessJournal of UrologyBladder Cancer: Upper Tract Transitional Cell Carcinoma I (PD43)1 Sep 2021PD43-04 A URINE-BASED DNA METHYLATION MARKER TEST TO DETECT UPPER TRACT UROTHELIAL CARCINOMA: A PILOT STUDY Alireza Ghoreifi, Sanam Ladi-Seyedian, Paolo Piatti, Benjamin Jara, Taikun Yamada, Lucy Sanossian, Sina Sobhani, Andrew Hung, Sumeet Bhanvadia, Rene Sotelo, Monish Aron, Mihir Desai, Inderbir Gill, Siamak Daneshmand, Gangning Liang, and Hooman Djaladat Alireza GhoreifiAlireza Ghoreifi More articles by this author , Sanam Ladi-SeyedianSanam Ladi-Seyedian More articles by this author , Paolo PiattiPaolo Piatti More articles by this author , Benjamin JaraBenjamin Jara More articles by this author , Taikun YamadaTaikun Yamada More articles by this author , Lucy SanossianLucy Sanossian More articles by this author , Sina SobhaniSina Sobhani More articles by this author , Andrew HungAndrew Hung More articles by this author , Sumeet BhanvadiaSumeet Bhanvadia More articles by this author , Rene SoteloRene Sotelo More articles by this author , Monish AronMonish Aron More articles by this author , Mihir DesaiMihir Desai More articles by this author , Inderbir GillInderbir Gill More articles by this author , Siamak DaneshmandSiamak Daneshmand More articles by this author , Gangning LiangGangning Liang More articles by this author , and Hooman DjaladatHooman Djaladat More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002057.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Upper tract urothelial carcinoma (UTUC) is an uncommon, yet lethal tumor of the urinary tract. Diagnosis and preoperative risk stratification of these patients present distinct challenges given the limitations of current available diagnostic tools, including endoscopic biopsy and imaging. Herein, we explore the feasibility of a urine-based epigenetic assay to detect UTUC. METHODS: Under an IRB approved protocol, urine samples were collected from UTUC patients before radical nephroureterectomy between December 2019 and December 2020. Patients with concomitant bladder cancer (n=4) and those who did not pass the quality control due to low DNA concentration (n=5) were excluded. Samples were analyzed with Bladder CARE™, a urine-based assay that measures methylation levels of 3 cancer biomarkers (TRNA-Cys, SIM2, and NKX1-1) and two internal control loci using methylation-sensitive restriction enzymes coupled with qPCR. Results are reported as Bladder CARE Index (BCI) score and categorized as positive (BCI>5), high-risk (BCI 2.5-5), or negative (BCI<2.5), which correlate to the concentration of cancer DNA in the sample. The findings were compared to 54 sex/age matched healthy (cancer-free) individuals. RESULTS: A total of 19 patients with a median age of 73 (14 male and 5 female) were included in the final analysis. Separate samples were sent from bladder and nephrostomy tube in 2 patients (total urine sample n=21). Final pathology revealed stage Ta (n=11), T1 (n=2), T2 (n=2), and T3 (n=4) UTUC. Among all samples, 20 showed positive and 1 high-risk BCI results. Urine cytology was available in 9 patients, in whom 3 had positive BCI results despite negative cytology. UTUC patients had a significantly higher BCI values compared to the control cases (average BCI value of 227.3 vs. 1.7, respectively; p < 0.0001) (Figure 1-A). The Receiver Operating Characteristic (ROC) curve demonstrating the sensitivity, specificity, positive predictive values, and negative predictive value of 100%, 87.1%, 75%, and 100%, respectively (AUC=0.996, Fig. 1B). CONCLUSIONS: In this pilot study, the proposed urine-based epigenetic assay showed high sensitivity and negative predictive value in the diagnosis of patients with UTUC. A larger sample size study to validate the accuracy of this test is the next step. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e731-e732 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alireza Ghoreifi More articles by this author Sanam Ladi-Seyedian More articles by this author Paolo Piatti More articles by this author Benjamin Jara More articles by this author Taikun Yamada More articles by this author Lucy Sanossian More articles by this author Sina Sobhani More articles by this author Andrew Hung More articles by this author Sumeet Bhanvadia More articles by this author Rene Sotelo More articles by this author Monish Aron More articles by this author Mihir Desai More articles by this author Inderbir Gill More articles by this author Siamak Daneshmand More articles by this author Gangning Liang More articles by this author Hooman Djaladat More articles by this author Expand All Advertisement Loading ...

Noble Metallic Pyramidal Substrate for Surface-Enhanced Raman Scattering Detection of Plasmid DNA Based on Template Stripping Method.

In this paper, a new method for manufacturing flexible and repeatable sensors made of silicon solar cells is reported. The method involves depositing the noble metal film directly onto the Si template and stripping out the substrate with a pyramid morphology by using an adhesive polymer. In order to evaluate the enhancement ability of the substrate, Rhodamine 6G (R6G) were used as surface-enhanced Raman scattering (SERS) probe molecules, and the results showed a high sensitivity and stability. The limit of detection was down to M for R6G. The finite-difference time domain (FDTD) was used to reflect the distribution of the electromagnetic field, and the electric field was greatly enhanced on the surface of the inverted pyramidal substrate, especially in pits. The mechanism of Raman enhancement of two types of pyramidal SERS substrate, before and after stripping of the noble metal film, is discussed. By detecting low concentrations of plasmid DNA, the identification of seven characteristic peaks was successfully realized using a noble metallic pyramidal substrate.

Aerobic fitness is associated with extracellular DNA levels in the sputum of patients with cystic fibrosis.

Patients with cystic fibrosis (CF) develop with progressive loss of lung function and aerobic fitness. However, the precise mechanisms of exercise intolerance are still controversial and appear to be influenced by several factors. This study aimed to evaluate the association of aerobic fitness with free DNA levels in the sputum of patients with CF. This cross-sectional study included patients with CF older than 6 years, free from active exacerbations, but who were able to produce spontaneously expectorated sputum. Extracellular DNA in the sputum was quantified. Lung function (spirometry) and aerobic fitness (cardiopulmonary exercise testing [CPET]) were performed. In addition, demographic, anthropometric and clinical data were collected. Sixteen patients with a mean age of 19.4±6.9 years and mean forced expiratory volume in the first second (FEV1 ) of 51.8±28.1 (% of predicted) were included. Mean peak oxygen consumption (VO2 peak) was 32.8±5.2 mL•kg-1 •min-1 , oxygen saturation at the end of the test was 90.6%±6.3% and mean extracellular DNA levels was 305.3±153.6 μg/mL. Individuals with a VO2 peak ≤ 30 mL•kg-1 •min-1 (P = .03) and a SpO2 ≤ 90% at the end of the test (P = .03) had a greater amount of extracellular DNA in the sputum. The proportion of patients with reduced VO2 peak in the group of patients with the lowest concentration of DNA in the sputum (<243 μg/mL) was significantly lower (0% vs 100%; P = .04). There is an association between the presence of free DNA in sputum and aerobic fitness in patients with CF.

Fish diversity monitoring in Maninjau Lake, West Sumatra using the eDNA with the next generation sequencing (NGS) technique

Various phenomena in nature and human activities have resulted in the loss of biodiversity, such as freshwater fish in Maninjau lake, West Sumatra, which has declined from year to year. A new method to monitor biodiversity quickly and efficiently is the NGS technique on environment DNA (eDNA). The study aimed to know the NGS technique’s ability and effectiveness to detect multiple species at one time from the water samples of Maninjau Lake. Water was taken from the surface as much as one L with two replications. The sequencing was used to identify species as shotgun metagenomic with universal primers. The results showed that 92 individuals were identified with 56 species from 24 genera, 16 families, and 12 orders. As much as 25% of fishes still cannot be grouped into the valid taxa (unclassified), which is allegedly related to the lack of available information database (NCBI) compared to the sequences obtained. NGS on the eDNA method detected two families (Cyprinidae and Cichlidae), three genera (Oreochromis, Cyprinus, and Xiphophorus), and two species of fish (Oreochromis niloticus and Cyprinus carpio) in Maninjau Lake, which were also previously reported using the conventional method. The native species were not detected in Maninjau lake by the eDNA method allegedly due to not being captured DNA from the collected water samples or the low DNA concentration, which cannot be continued to the PCR process. Thus, several efforts are needed to reduce the limitations in monitoring using the eDNA method to obtain the maximal results. The eDNA method can be a useful tool for monitoring biodiversity. The result can determine the conservation strategies, especially for the fishes in Maninjau Lake, West Sumatra.

Differences of Human Cytomegalovirus Strains in Recipients of Allogeneic Hematopoietic Stem Cells before and after Transplantation

Background. Human cytomegalovirus (HCMV) is a ubiquitous viral infection of worldwide distribution. This virus is the most significant infectious cause of congenital disease, an important opportunist in the immunocompromised hosts, such as allogeneic hematopoietic stem cells transplantation (allo-HSCT) recipients (Mokarski E.S.,2013). If seropositive recipient develops HCMV infection after allo-HSCT, it is usually unknown whether it is a reactivation of endogenous HCMV or a reinfection by a new exogenous HCMV strain. The impact of genetic diversity of HCMV on its pathogenesis has not been well evaluated by now. However, the emerging data indicate a possible correlation: the viral load and the course of the infection were different in AIDS patients with different HCMV genotypes (Renzette, 2015). The estimation of a possible link between viral genotype and probability of HCMV infection occurrence is relevant. Very little data on difference in HCMV genotypes within one patient before and after allo-HSCT is available.The aim. The aim of this study was the identification of difference in HCMV UL139 gene sequences before and after the allo-HSCT.Materials and Methods. Patients: 13 allo-HSCT recipients (5 ALL, 3 AML, 3 CML, 1 AA, 1 lymphoma of the mantle) were enrolled in the study, male/female 6/7; median age was 27 (22-49) years. Patients' blood samples were collected within 6 month before and 6 month after transplantation and tested for the HCMV DNA by PCR. Positive samples were sequenced by Sanger inside the UL139 (putative membrane gp gene) region for genotyping. Sequences within one patient before and after transplantation were compared by BLAST (Basic Local Alignment Search Tool) software.Results and discussion. Sequencing was successfully completed only in samples of 7 patients (pts), probably due to low concentration of the viral DNA in the samples or other technical issues. The comparison of sequences showed (see table) that 3 out of 7 pts had reactivation of endogenous virus, because sequences exhibited 100% match. Another 2 out of 7 patients' sequences showed about 88% match. This indicates change in genotype to phylogenetically closest one. The rest 2 pts did not show any similarity in sequences at all which definitely points to reinfection after allo-HSCT. Data obtained are similar to a recent study by Zawilinska (Zawilinska B, 2016), which appeals that reinfection occurs slightly more often than reactivation. Data analys showed that reactivation was detected in patients with AML, reinfection in patients with ALL. Average period before reactivation occurrence was 27 days and before reinfection occurrence - 111 days. Statistical reliability cannot be obtained due to patients' number paucity. The genotyping of patients' HCMV by comparison of sequences with known genotypes of the UL139 gene from GenBank® revealed that before transplantation HCMV genotype in 3 patients had a strong resemblance to genotype 2, another 2 patients had genotype close to the genotype 5, and 1 patient had genotype 1. After transplantation genotype 2 became dominant and was identified in 6 out of 7 patients (see table).Conclusions. The conducted study shows that HCMV infection after allo-HSCT may be the result of both reactivation of endogenous virus and reinfection by exogenous strain. Reactivation was detected in patients with AML only, reinfection - in patients with ALL only. Reactivation developed earlier than reinfection (27 days versus 111 days). The chosen method allowed us to detect several mononucleotide substitutions in sequences, what can be useful in cases when no evident change of genotype happened after allo-HSCT. There are several possible explanations of the occurrence of reinfection: possible transmission from the outer environment or reactivation of a minor genotype if 2 or more HCMV genotypes originally had taken place. Further studies are required for more reliable conclusions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DNA Minor Groove-Induced cis-trans Isomerization of a Near-Infrared Fluorescent Probe.

The discovery of small molecules that exhibit turn-on far-red or near-infrared (NIR) fluorescence upon DNA binding and understanding how they bind DNA are important for imaging and bioanalytical applications. Here we report the DNA-bound structure and the DNA binding mechanism of quinone cyanine dithiazole (QCy-DT), a recently reported AT-specific turn-on NIR fluorescent probe for double-stranded DNA. The nuclear magnetic resonance (NMR)-derived structure showed minor groove binding but no specific ligand-DNA interactions, consistent with an endothermic and entropy-driven binding mechanism deduced from isothermal titration calorimetry. Minor groove binding is typically fast because it minimally perturbs the DNA structure. However, QCy-DT exhibited unusually slow DNA binding. The cyanine-based probe is capable of cis-trans isomerization due to overlapping methine bridges, with 16 possible slowly interconverting cis/trans isomers. Using NMR, density functional theory, and free energy calculations, we show that the DNA-free and DNA-bound environments of QCy-DT prefer distinctly different isomers, indicating that the origin of the slow kinetics is a cis-trans isomerization and that the minor groove preferentially selects an otherwise unstable cis/trans isomer of QCy-DT. Flux analysis showed the conformational selection pathway to be the dominating DNA binding mechanism at low DNA concentrations, which switches to the induced fit pathway at high DNA concentrations. This report of cis/trans isomerization of a ligand, upon binding the DNA minor groove, expands the prevailing understanding of unique discriminatory powers of the minor groove and has an important bearing on using polymethine cyanine dyes to probe the kinetics of molecular interactions.

DNA-encoded bimetallic Au-Pt dumbbell nanozyme for high-performance detection and eradication of Escherichia coli O157:H7

Infectious Escherichia coli O157:H7 threatens the health of millions people each year. Thus, it is important to establish a simple and sensitive method for bacterial detection and eradication. Herein, a DNA-programming strategy is explored to synthesize anisotropic dumbbell-like Au-Pt nanoparticles with excellent catalytic and anti-bacterial activities, which were applied in the simultaneous detection and eradication of pathogenic bacteria. The DNA sequence-dependent growth of bimetallic nanoparticles is firstly studied and polyT20 has the tendency to form dumbbell-like Au-Pt bimetallic structures based on gold nanorods seeds. PolyA20 and polyC20 can also form similar structures but only at much lower DNA concentrations, which can be explained by their much higher affinity to the metal surfaces than T20. The as-prepared nanoparticles exhibit high nanozyme catalytic activity resulting from the synergistic effect of Au and Pt. Under light irradiation, the Au-Pt nanoparticles show high photothermal conversion efficiency and enhanced catalytic activity, which can be applied for the eradication and detection of E. coli O157:H7 with a robust efficacy (95%) in 5 min and provides excellent linear detection (10–107 CFU/mL), with a detection limit of 2 CFU/mL. This study offered new insights into DNA-directed synthesis of nanomaterials with excellent biosensing and antibiotic applications.