Presence Of DNA Sequences Research Articles

Rapid SARS-CoV-2 sensing through oxygen reduction reaction catalysed by Au@Pt/Au core@shell nanoparticles

The development of rapid, accurate, sensitive, and low-cost diagnostic methods for COVID-19 detection in real-time is the unique way to control infection sources and monitor illness progression. In this work, we propose an electrochemical biosensor for the rapid and accuracy diagnosis of COVID-19, through the determination of ORF1ab specific sequence. The biosensor is based on the immobilization of a thiolated sequence partially complementary (domain 1) to ORF1ab on gold screen-printed electrodes and the use of bifunctional Au@Pt/Au core@shell nanoparticles modified with a second thiolated sequence partially complementary to ORF1ab (domain 2) as electrochemical indicator of the hybridization of DNA sequences. The synthesized Au@Pt/Au nanoparticles consist of an Au core, a shell of Pt (Au@Pt NPs), that provides an excellent electrocatalytic activity toward the oxygen reduction reaction (ORR) even after formation of hybrid biomaterials by modification, through the Au protuberances growth on the NPs surface, with an oligonucleotide with recognition ability. The ORR electrochemical activity, enhanced by the label element (Au@Pt/Au NPs), has been employed, for the first time, as indicator of the hybridization event. Based on this strategy, target sequences of the SARS-CoV-2 virus have been detected with a detection limit of 32 pM. The selectivity of the biosensor was confirmed by analysing ORF1ab sequence in the presence of DNA sequences from other viruses. The biosensor has been successfully applied to the direct detection of the virus in non-amplified samples of nasopharyngeal swabs from infected and non-infected patients. Results compare well with those obtained through RT-qPCR but our method is more rapid since does not need any amplification process.

Here, there and everywhere: Ecology and biology of the Dependentiae phylum.

Our view of bacterial diversity has been dramatically impacted by cultivation-independent approaches such as metagenomics and 16S rRNA gene sequencing. Consequently, most bacterial phyla known to date are only documented by the presence of DNA sequences in databases and lack cultivated representatives. This bacterial majority that is yet-to-be cultivated, is forming the 'Microbial Dark Matter', (MDM) a consortium, whose ecology and biology remain largely unexplored. The Candidatus Dependentiae stands as one of many phyla within this MDM, found worldwide in various environments. Genomic evidence suggests ancestral, unusual adaptations of all Ca. Dependentiae to a host dependent lifestyle. In line with this, protists appear to be important for Ca. Dependentiae biology, as revealed by few recent studies, which enabled their growth in laboratory through host cultivation. However, the Ca. Dependentiae still remain to this day a poorly documented phylum. The present review aims to summarize the current knowledge accumulated on this often found, but rarely highlighted, bacterial phylum.

Role of clonoSEQ®, a Next-Generation Sequencing (NGS) Assay and PET/CT As a Measure of Minimal Residual Disease Negativity Among Patients with Multiple Myeloma

Role of clonoSEQ®, a Next-Generation Sequencing (NGS) Assay and PET/CT As a Measure of Minimal Residual Disease Negativity Among Patients with Multiple Myeloma

Visual detection of G-quadruplex with mushroom derived highly fluorescent carbon quantum dots

G-quadruplexes are secondary DNA structures frequently found in telomeres and DNA sequences related to gene regulation, etc. Herein, we report a label free, sensitive and visually detectable fluorescence based biosensing platform for the detection of G-quadruplexes in DNA. Highly fluorescent N-doped carbon quantum dot (CD) with enriched functional groups was synthesized from Ganoderma lucidum, an oriental fungus by using a green hydrothermal process. Noncovalent functionalization of CD was done with Hemin where Hemin/CD conjugate forms a quenched union. However, in presence of DNA sequences that contain G-quadruplexes, the fluorescence of CD is restored selectively. The fluorescence restoration of CD is attributed to the stripping of Hemin from Hemin/CD conjugate that preferably slide into the G quadruplexes leaving CD. This increase in CD fluorescence is easily detectable under hand-held UV light without any other instrumental intervention for G-quadruplex containing DNA only and not in any other DNA samples. Also, such selective fluorescent recovery was not observed with chemically synthesized CD that lack hydroxyl functionality.

The blood DNA virome in 8,000 humans.

The characterization of the blood virome is important for the safety of blood-derived transfusion products, and for the identification of emerging pathogens. We explored non-human sequence data from whole-genome sequencing of blood from 8,240 individuals, none of whom were ascertained for any infectious disease. Viral sequences were extracted from the pool of sequence reads that did not map to the human reference genome. Analyses sifted through close to 1 Petabyte of sequence data and performed 0.5 trillion similarity searches. With a lower bound for identification of 2 viral genomes/100,000 cells, we mapped sequences to 94 different viruses, including sequences from 19 human DNA viruses, proviruses and RNA viruses (herpesviruses, anelloviruses, papillomaviruses, three polyomaviruses, adenovirus, HIV, HTLV, hepatitis B, hepatitis C, parvovirus B19, and influenza virus) in 42% of the study participants. Of possible relevance to transfusion medicine, we identified Merkel cell polyomavirus in 49 individuals, papillomavirus in blood of 13 individuals, parvovirus B19 in 6 individuals, and the presence of herpesvirus 8 in 3 individuals. The presence of DNA sequences from two RNA viruses was unexpected: Hepatitis C virus is revealing of an integration event, while the influenza virus sequence resulted from immunization with a DNA vaccine. Age, sex and ancestry contributed significantly to the prevalence of infection. The remaining 75 viruses mostly reflect extensive contamination of commercial reagents and from the environment. These technical problems represent a major challenge for the identification of novel human pathogens. Increasing availability of human whole-genome sequences will contribute substantial amounts of data on the composition of the normal and pathogenic human blood virome. Distinguishing contaminants from real human viruses is challenging.

Genomic and proteomic characterization of “Candidatus Nitrosopelagicus brevis”: An ammonia-oxidizing archaeon from the open ocean

Thaumarchaeota are among the most abundant microbial cells in the ocean, but difficulty in cultivating marine Thaumarchaeota has hindered investigation into the physiological and evolutionary basis of their success. We report here a closed genome assembled from a highly enriched culture of the ammonia-oxidizing pelagic thaumarchaeon CN25, originating from the open ocean. The CN25 genome exhibits strong evidence of genome streamlining, including a 1.23-Mbp genome, a high coding density, and a low number of paralogous genes. Proteomic analysis recovered nearly 70% of the predicted proteins encoded by the genome, demonstrating that a high fraction of the genome is translated. In contrast to other minimal marine microbes that acquire, rather than synthesize, cofactors, CN25 encodes and expresses near-complete biosynthetic pathways for multiple vitamins. Metagenomic fragment recruitment indicated the presence of DNA sequences >90% identical to the CN25 genome throughout the oligotrophic ocean. We propose the provisional name "Candidatus Nitrosopelagicus brevis" str. CN25 for this minimalist marine thaumarchaeon and suggest it as a potential model system for understanding archaeal adaptation to the open ocean.

Identifikace a charakterizace bakterií rodu Enterococcus určených pro výrobu funkčních potravin

Cilem teto prace byla rodova a druhova identifikace dvanacti probiotických kmenů rodu Enterococcus ze Sbirky cistých mlekařských kultur Laktoflora (CCDM, Milcom Praha, Ceska republika). K identifikaci byla použita polymerazova řetězova reakce (Dutka-Malen a kol, 1995). Vsechny analyzovane kmeny byly zařazeny do rodu Enterococcus a druhu E. faecium. U vsech kmenů byly prokazany některe probioticke vlastnosti a mohly by být použity pro výrobu funkcnich potravin. Vzhledem ke skutecnosti, že byly některe kmeny E. faecium ve fermentovaných potravinach popsany jako producenti biogennich aminů, byly kmeny analyzovane v teto praci prověřeny na přitomnost ci nepřitomnost genů pro tyrosindekarboxylazu a histidindekarboxylazu. Pro skrining byla použita PCR popsana Cotonem a kol., 2004. Přitomnost genu pro histidindekarboxylazu nebyla potvrzena v žadnem z analyzovaných kmenů E. faecium, přitomnost genu pro tyrosindekarboxylazu byla potvrzena u vsech kmenů podrobených skriningu. Při aplikaci testovaných kmenů E. faecium jako startovacich kultur pro výrobu fermentovaných mlecných výrobků musi být ve finalnich výrobcich a po dobu jejich uchovavani testovana produkce biogenniho aminu tyraminu.

Abstract 3172: Detection of viral HPV DNA in sporadic colorectal cancers in relation to CpG island methylator phenotype (CIMP)

Abstract Background: There is evidence that insertion of viral DNA into a mammalian genome can lead to alterations of methylation patterns. To test the possible correlation between epigenetic modulations in colorectal cancer (CRC) and HPV infection, we conducted a MSP- PCR methylation analysis. The aim of the present study was to examine the presence of DNA sequences of human papillomavirus (HPV) in CRC and correlate this data with their CIMP status. Methods: Thirty-seven colorectal adenocarcinomas were screened for HPV infection using nested-PCR (PGMY09/11-GP5+/6+). HPV typing was performed by type-specific PCR for HPV 16 and HPV 18 DNA. Eight CpG island methylator phenotype (CIMP) genes specific to sporadic colorectal cancer (MLH1, CACNA1G, NEUROG1, IGF2, SOCS1, RUNX3, CDKN2A and CRABP1) were assessed by methylation-specific PCR. Results: Thirty-seven CRC cases (mean age at diagnosis 63.2 ± 11.7 years; 17 males) were evaluated. Tumors were mostly located in the left colon (73.0%), and moderate differentiated (61.1%). HPV's DNA was identified in 23 of 37 (62.2%) CRC studied cases. CRABP1 (59.5%), CDKN2A (37.8%), MLH1 (13.5%), and NEUROG1 (10.8%) were the most commonly methylated genes with a discrete clinic-pathological phenotype and gene methylation pattern. No statistical associations between HPV status and methylation patterns were found, after correlation with tumor staging, cell differentiation, tumor location, and life style. Discussion: We reported that the CIMP-hypermethylation may be less frequent among Puerto Ricans Hispanics than previously reported in other racial and/or ethnic groups. However, a correlation could not be established (p>0.5) between HPV infection and the aberrant methylation pattern in the genes panel. We observed the trend that HPV is more prevalent in CRC without aberrant methylation patterns. This might suggest that hypermethylation could be a protective factor for HPV infection. This inference may mean that HPV positive colorectal cancer could represent a distinctive clinical and epidemiological condition compared with HPV-negative CRC. Citation Format: Raul D. Bernabe-Dones, Wesley Villavicencio-Torres, Cristina Munoz-Masso, Yaritza Reyes -Medina, Lorena Marcano-Bonilla, Hector Perez-Cantalapiedra, Sharon Fonseca-Williams, Mercedes Y. Lacourt-Ventura, Yasuhiro Yamamura, Nayra Rodriguez, Marcia R. Cruz-Correa. Detection of viral HPV DNA in sporadic colorectal cancers in relation to CpG island methylator phenotype (CIMP). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3172. doi:10.1158/1538-7445.AM2014-3172

Abstract 743: Pu-27 G-quadruplex induces uncompensated DNA damage response by destabilizing shelterin complex at telomere and non-telomeric region leading to cell death.

Abstract Throughout the genome as well as telomere region, there is presence of DNA sequences which forms quadruplex. Here we show Pu-27 triggers the damage of cells by interfering telomeric ‘shelterin’ protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks (DSB). We found that Pu-27 frequently prompted chromosomal aberrations including abnormal metaphases (45%), chromatid breaks (40%) chromosomal break (27%) by karyotyping of U937 metaphase cells and was associated with induction of DNA damage response sensor γ-H2AX. Analysis of DNA damage response regulators by RT-PCR array of Pu-27 treated U937 cells revealed downregulation of telomeric ‘shelterin’ proteins (TRF2, TRF1, POT1 and TIN2), upstream kinase ATM, DNA damage response mediators (RAD17, RAD50 and 53BP1), and cell cycle arrest molecules (CHK1 and CHK2). Interestingly, there were no changes or low expression of DNA repair molecules (H2AX and BRCA1) and telomere maintenance gene TERT respectively. αB-U937 cell, where basic-domain of shelterin protein TRF2 was deleted became relatively resistance to Pu-27 and showed no changes of constitutively active form of γ-H2AX. RT-PCR array of Pu-27 treated αB-U937 cell appeared to be no changes of telomeric ‘shelterin’ proteins (TRF2, TRF1, POT1 and TIN2), DNA damage response mediators (RAD17, RAD50 and 53BP1); down regulation of upstream kinase ATM; up regulation of cell cycle arrest molecules (SMC1, CHK1 and CHK2), DNA repair molecules (H2AX and BRCA1), and telomere maintenance gene TERT. Shelterin protein, TRF1 co-associated with γH2AX by inducing TIFs (Telomere-dysfunction Induced Foci) when U937 cell was treated with Pu-27. Sk-Lu-1, an Alt (Alternating Lengthening to Telomere) cell which does not use TERT for their growth and propagation is relatively resistance to Pu-27. ATM preserves the telomeric homeostasis by maintaining the integrity of ‘shelterin’ protein. To substantiate the specificity of Pu-27 to shelterin protein complex, we found ATM-deficient cell was more sensitive to Pu-27 than ATM-proficient cell. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, these results suggest that Pu-27-treated sensitivity at least in part due to compromising telomeric shelterin complex and perhaps stabilizing telomeric quadruplex and not by directly inhibiting telomerase enzyme. U937 cell with chromosomal aberration and unstable telomere progress through cell cycle without arrest and repair leading to cell death whereas αB-U937 cells is continuously repaired by arresting and induction of repair machinery. Continuous exposure of Pu-27 results in refractory to repair of DNA damage leading to uncompensated genomic stability leading to cell death. Citation Format: Md Ashraful Islam, Shelia D. Thomas, Kara J. Sedoris, Donald M. Miller. Pu-27 G-quadruplex induces uncompensated DNA damage response by destabilizing shelterin complex at telomere and non-telomeric region leading to cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2013-743 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

DNA Sequence-Enabled Reassembly of the Green Fluorescent Protein

We describe a general methodology for the direct detection of DNA by the design of a split-protein system that reassembles to form an active complex only in the presence of a targeted DNA sequence. This approach, called SEquence Enabled Reassembly (SEER) of proteins, combines the ability to rationally dissect proteins to construct oligomerization-dependent protein reassembly systems and the availability of DNA binding Cys2-His2 zinc-finger motifs for the recognition of specific DNA sequences. We demonstrate the feasibility of the SEER approach utilizing the split green fluorescent protein appended to appropriate zinc fingers, such that chromophore formation is only catalyzed in the presence of DNA sequences that incorporate binding sites for both zinc fingers.

Defining the anatomy of the Rangifer tarandus sex chromosomes.

A comprehensive cytogenetic characterization of the unusally large reindeer (Rangifer tarandus) sex chromosomes is presented for the purpose of studying the evolution of these atypical gonosomes. Sex chromosome idiograms were constructed from G-banded and C-banded chromosomes to illustrate the relative amounts and locations of euchromatin and heterochromatin. Hybridization with a Mazama gouazoubira X whole-chromosome paint revealed that essentially all reindeer X-linked euchromatin and most reindeer Y-linked euchromatin is conserved interspecifically. Subsequently, painting probes were generated from flow-sorted reindeer X chromosomes, flow-sorted reindeer Y chromosomes, and from microdissections of specific gonosomal regions to establish specific segment-to-segment homologies between these gonosomes. In particular, one microdissection-generated paint demonstrated that certain constituent repetitive DNAs, found in C-band region Xq31, were also present in essentially all heterochromatin blocks of the Y chromosome. Microdissection-generated paints from other X-linked heterochromatin blocks revealed the presence of DNA sequences that lacked homologous sequences on the Y chromosomes and were more specific for their region of origin. These characteristics of the reindeer sex chromosomes are consistent with the notion that mammalian sex chromosomes were derived from homologous progenitor chromosome pairs and provide insights into the evolution of these atypical mammalian gonosomes.

Influence of DNA sequence identity on efficiency of targeted gene replacement.

We have developed a system for analyzing recombination between a DNA fragment released in the nucleus from a single-copy plasmid and a genomic target in order to determine the influence of DNA sequence mismatches on the frequency of gene replacement in Saccharomyces cerevisiae. Mismatching was shown to be a potent barrier to efficient gene replacement, but its effect was considerably ameliorated by the presence of DNA sequences that are identical to the genomic target at one end of a chimeric DNA fragment. Disruption of the mismatch repair gene MSH2 greatly reduces but does not eliminate the barrier to recombination between mismatched DNA fragment and genomic target sequences, indicating that the inhibition of gene replacement with mismatched sequences is at least partially under the control of mismatch repair. We also found that mismatched sequences inhibited recombination between a DNA fragment and the genome only when they were close to the edge of the fragment. Together these data indicate that while mismatches can destabilize the relationship between a DNA fragment and a genomic target sequence, they will only do so if they are likely to be in the heteroduplex formed between the recombining molecules.

Structure, Sequence, and Chromosome 19 Localization of HumanUSF2and Its Rearrangement in a Patient with Multicystic Renal Dysplasia

The precise etiology of hydronephrosis caused by pelvi-ureteric junction obstruction is not yet known but there is convincing evidence for a genetic cause, with linkage analysis predicting a hereditary hydronephrosis locus on chromosome 6p. In previous studies, a patient was described with ade novoautosomal t(6;19)(p21;q13.1) translocation and suffering from bilateral multicystic renal dysplasia (MRD) caused by a bilateral complete pelvi-ureteric junction obstruction. In an effort to elucidate a possible correlation between this translocation and hereditary hydronephrosis, we have carried out an extensive molecular characterization of a chromosome 19 cosmid clone previously identified as spanning the translocation in this unique index case. DNA sequencing across a 9.2-kbBamHI fragment that straddles the translocation indicates the presence of DNA sequences with a high degree of similarity to theUSF2gene that encodes the transcription factor USF2 (upstream stimulator factor 2). The genomic structure ofUSF2consists of 10 exons distributed over a DNA region of about 11 kb. The putative promoter region is GC-rich and lacks TATA and CCAAT boxes, suggesting that expression of theUSF2gene may be controlled by a typical housekeeping gene promoter. The chromosome 19 breakpoint in the MRD patient appeared to have occurred in intron 7 of theUSF2gene. Northern blot analysis of a variety of human tissues revealed that theUSF2gene is ubiquitously expressed. Furthermore, Northern blot and 3′-RACE analysis of mRNA isolated from lung fibroblasts of the MRD patient failed to detect a fusion transcript involvingUSF2sequences, suggesting gene disruption rather than the generation of a fusion gene as a possible underlying mechanism.

AIDS-related non-Hodgkin's lymphomas: molecular genetics, viral infection and cytokine deregulation.

AIDS-related non-Hodgkin's lymphomas (AIDS-NHLs) are almost invariably derived from B cells and are grouped into three distinct histologic categories, including small-non-cleaved-cell lymphoma (SNCCL), diffuse large-cell lymphoma (DLCL), and anaplastic large-cell lymphoma (ALCL). In addition, AIDS-NHLs presenting solely as a body cavity effusion are thought to be a peculiar clinicopathologic entity and are defined as body-cavity-based lymphoma (BCBL). At the biologic level, AIDS-related lymphomagenesis is characterized by activation of proto-oncogenes, inactivation of tumor suppressor genes, viral infection of the tumor clone, and deregulated cytokine production. Distinct AIDS-NHL types associate with specific molecular pathways. The first pathogenetic pathway clusters with AIDS-SNCCL, and is characterized by a relatively mild degree of host immunodeficiency. AIDS-SNCCL consistently associates with c-myc rearrangements and p53 inactivation in 100 and 60% of cases, respectively, whereas infection by Epstein-Barr virus (EBV) is restricted to 30% of the cases. Production of high levels of interleukin-10 is an additional peculiar feature of EBV-positive AIDS-SNCCL. The second pathogenetic pathway associates with AIDS-DLCL, which is usually accompanied by marked host immunodeficiency. AIDS-DLCL is characterized by EBV infection in the large majority of cases and by the mutually exclusive presence of bcl-6 rearrangements and c-myc translocations in 40% of the cases. A third pathway characterizes AIDS-BCBL, which associates in virtually all cases with infection by EBV and with the presence of DNA sequences of the recently identified Kaposi sarcoma herpesvirus in the apparent absence of other known genetic lesions. Finally, the pathogenetic features of AIDS-ALCL are still under investigation.

Prevalence of HLA-DQ beta chain non-asp alleles in type I (insulin-dependent) diabetics with young and older ages of onset

Particular HLA-DQ beta chain alleles were reported as immunogenetic markers of type I diabetes mellitus with young onset of the disease. In a homogeneous German population, we studied HLA-DR specificities and HLA-DQ beta chain alleles in young-onset (less than 21 years of age; n = 185) and adult-onset (greater than 40 years of age; n = 48) insulin-dependent diabetics. In both cohorts of type I diabetics, the HLA-DR3 and -DR4 specificities were significantly increased. The presence of an HLA haplotype with an amino acid other than aspartic acid at position 57 of the DQ beta chain was significantly associated with type I diabetes in both cohorts (etiologic fraction: 93% and 73%). We conclude that the presence of DNA sequences coding for an amino acid other than aspartic acid at the 57th position of the DQ beta chain provides a molecular risk marker for type I diabetes of both and adult onset.

δ-like haemolysin produced by Staphylococcus lugdunensis

Thirty independent clinical isolates of Staphylococcus lugdunensis were tested for expression of haemolysin characteristics of Staphylococcus aureus and for the presence of DNA sequences with homology to S. aureus toxin genes. Twenty-six strains produced a thermostable haemolysin with greatest activity against rabbit erythrocytes. DNA-DNA hybridisation was performed with S. lugdunesis DNA and probed with the alpha, beta, delta and gamma toxin gene probes of S. aureus as well as that for coagulase. Hybridisation was found with the delta probe in 28 strains including 6 which did not express haemolysin. Two strains did not hybridise with any of the probes. These findings suggest that S. lugdunensis strains produce a delta-like toxin which shares homology with the delta toxin of S. aureus.

Spontaneous deletion of a 20-kilobase DNA segment carrying genes specifying isopropylbenzene metabolism in Pseudomonas putida RE204.

The genes encoding isopropylbenzene metabolism in Pseudomonas putida RE204 are readily lost in two ways: by loss (curing) of plasmid pRE4 which specifies the catabolic pathway and by deletion from pRE4 of an approximately 20-kilobase segment of DNA carrying the catabolic genes. The presence of DNA sequences at the ends of the catabolic gene region sharing homology with one another suggests that the deletions result from recombination events between these homologous sequences.

Distribution of DNA sequences homologous to nifH among archaebacteria

Using the Southern hybridization technique, 22 strains of archaebacteria were tested for the presence of DNA sequences homologous to theAnabaena nifH gene, which codes for the Fe protein component of nitrogenase. Total DNA from all the 14 methanogenic strains tested was shown to carry homology tonifH. With total DNA from 3Halobacterium strains, hybridization was also detected, though unspecific binding cannot completely be excluded. No hybridization was detected with DNA fromThermoplasma acidophilum, nor with the DNA from 4 strains belonging to the sulfur-dependent branch of the archaebacteria. Results strongly support the hypothesis of an ancient origin ofnif genes.