Relationships of mitochondrial DNA mutations and select clinical diagnoses in perinatally HIV- and ART-exposed uninfected children.

The prevalence of pathogenic mutations within mitochondrial (mt) DNA of youth who were perinatally exposed to HIV and ART but remained uninfected (YHEU) were assessed relative to phenotypic clinical indicators of mitochondrial dysfunction (MtD). This was a cross-sectional, nested case-control study. A total of 144 cases met at least one clinical MtD definition and were matched with up to two controls each (n = 287). At least one risk mutation was present in nearly all YHEU (97 %). No differences in mutation frequencies were observed between metabolic or neurodevelopmental cases and respective controls; however, higher frequencies were found in controls versus respective neurologic or growth cases.

Study on identification of medicinal plant Gentiana scabra Bge. in Liaoning province based on DNA barcode sequences / 国际中医中药杂志

Objective:To identify and analyze the genuine medicinal plant Gentiana scabra Bge. from 9 regions in Liaoning Province using DNA barcode technology based on the base sequence of internal transcribed spacer. Methods:DNA was extracted from the medicinal parts of 26 Gentiana scabra Bge. samples by using DNA kit extraction method. The ITS sequence was amplified through polymerase chain reaction (PCR), and then two-way sequencing was carried out. Other sources and outgroup sequences of the medicinal plant Gentiana scabra Bge. were downloaded from Genbank. After the sequencing results were spliced by using SeqMan 7.1.0 software, MEGA 7.0 software was used to analyze and compare the data, and calculate the genetic distance of K2P (Kimura 2-parameter). The phylogenetic tree was established by Neighbor-Joining (NJ) method for analysis. Results:According to the results of NJ cluster tree, all Gentiana scabra Bge. samples from different sources were clustered into one large branch, and Gentiana scabra Franch. and Gentiana triflora Pall. were clustered into one branch respectively, with obvious differences; Gentiana scabra and Gentiana manshurica Kitag. were clustered into one branch, and the genetic relationship was relatively close. In combination with the variation site and genetic distance, the base sequences of Gentiana scabra and Gentianamanshurica were very similar, and the interspecific differences were very small. Except for the intraspecific variation of only one sample collected in Liaoning Province, the base sequences of the other samples were the same, and there was no difference between " Gentiana scabra Bge. in Qingyuan" and Gentiana scabra Bge. samples from other regions in Liaoning Province. Conclusion:The DNA barcode technology of ITS sequence can be used to differentiate and identify medicinal plant Gentiana scabra Bge. and its original plants from different sources with a high success rate.

A Case Report of Rubinstein-Taybi Syndrome Presenting with Extensive Keloid Formation and Review of Literature.

Rubinstein-Taybi syndrome (RSTS) is an extremely rare genetic disorder affecting multi-organ systems. A tendency to form keloid is one of the common dermatologic manifestations. We describe a 23-year-old female presented with extensive keloids which developed spontaneously. She had typical facial features, broad thumbs, and dental defects, which were suspicious features of genetic syndrome. Direct sequencing for cyclic-AMP-regulated enhancer binding protein revealed a novel mutation. So far, 23 cases of RSTS have been reported in Korean literature. To the best of our knowledge, this is the first report in Korea to describe confirmed case of RSTS with extensive keloids as a chief manifestation.

Discovery of a tRNA-like base sequence in the coronavirus genome and possible mechanism of action.

BACKGROUND: The question of whether the coronavirus genome contain as-yetununderstood genetic component. PURPOSE (OBJECTIVE): Elucidate the novel functions of the discovered tRNA-like base sequence and lead to the development of novel therapeutic agents. METHODS: A novel tRNA-like base sequence was found in the sequences complementary to the genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV. By comparing mutations in the tRNA-like base sequences of these two viruses, it was found that base pairing in the cloverleaf model of SARS-CoV-2 was more robust than that of SARS-CoV. RESULTS: The results of homology search between a short sequence of the coronavirus tRNA-like base sequence and human genes suggest that the molecule produced by this novel tRNA-like sequence may be involved in the splicing of human messenger RNA. CONCLUSIONS: Experimental molecular evidence of the tRNA-like base sequence discovered in this study is urgently needed.

Interaction of Proteins with Inverted Repeats and Cruciform Structures in Nucleic Acids.

Cruciforms occur when inverted repeat sequences in double-stranded DNA adopt intra-strand hairpins on opposing strands. Biophysical and molecular studies of these structures confirm their characterization as four-way junctions and have demonstrated that several factors influence their stability, including overall chromatin structure and DNA supercoiling. Here, we review our understanding of processes that influence the formation and stability of cruciforms in genomes, covering the range of sequences shown to have biological significance. It is challenging to accurately sequence repetitive DNA sequences, but recent advances in sequencing methods have deepened understanding about the amounts of inverted repeats in genomes from all forms of life. We highlight that, in the majority of genomes, inverted repeats are present in higher numbers than is expected from a random occurrence. It is, therefore, becoming clear that inverted repeats play important roles in regulating many aspects of DNA metabolism, including replication, gene expression, and recombination. Cruciforms are targets for many architectural and regulatory proteins, including topoisomerases, p53, Rif1, and others. Notably, some of these proteins can induce the formation of cruciform structures when they bind to DNA. Inverted repeat sequences also influence the evolution of genomes, and growing evidence highlights their significance in several human diseases, suggesting that the inverted repeat sequences and/or DNA cruciforms could be useful therapeutic targets in some cases.

Molecular factors governing perineural invasion in malignancy.

BACKGROUND: Perinueral invasion (PNI) is recognized as an independent adverse prognostic factor associated with shorter disease free and disease specific survival in a range of malignancies. However, not all histologically detected PNI demonstrate aggressive biologic behaviour. Herein, we systematically review the literature to identify neurotrophic biomarkers that may potentially be used to predict the biologic potential of PNI. METHOD: A systematic review was conducted based on PRISMA guidelines utilising the search terms 'PNI', 'DNA' and 'RNA' analysis in select malignancies following registry of the search strategy on PROSPERO. The biologic role of the molecular markers identified through the literature review was examined using publicly available databases, such as Gene Cards and Kyoto Encyclopedia of Genes and Genomes (KEGG) with a focused literature review of the identified pathways. RESULTS: The systematic search identified 256 studies, of which 78 studies were suitable for data extraction. A variety of methodologies including immunohistochemistry, immunoblotting, nucleic acid sequencing, Luciferase assays and CRISPR techniques have been undertaken to evaluate the biologic potential of PNI. The studies evaluated 136 unique molecules. Of these, only 15 molecules were investigated through multiple studies with concordant results or had robust functional analyses. Three pathways were identified as playing a role in PNI, namely; the epithelial-mesenchymal transition pathway, neurotrophic pathway and Notch pathway. DISCUSSION: Our understanding of the complex and reciprocal interaction between tumour and nerve cells that drives PNI is still evolving. The knowledge gaps can largely be attributed to publication bias, lack of availability of high-quality patient derived tissues and limitations of currently available technology. This review summarises the current knowledge regarding development and progression of PNI that can be harnessed for prognostication and treatment. This review also summarises the lacunae in our understanding of the pathogenesis of PNI thus identifying avenues for future studies.

Impacts of Molecular Structure on Nucleic Acid-Protein Interactions.

Interactions between nucleic acids and proteins are some of the most important interactions in biology because they are the cornerstones for fundamental biological processes, such as replication, transcription, and recombination [...].

Evolution of Diverse Strategies for Promoter Regulation.

DNA is fundamentally important for all cellular organisms due to its role as a store of hereditary genetic information. The precise and accurate regulation of gene transcription depends primarily on promoters, which vary significantly within and between genomes. Some promoters are rich in specific types of bases, while others have more varied, complex sequence characteristics. However, it is not only base sequence but also epigenetic modifications and altered DNA structure that regulate promoter activity. Significantly, many promoters across all organisms contain sequences that can form intrastrand hairpins (cruciforms) or four-stranded structures (G-quadruplex or i-motif). In this review we integrate recent studies on promoter regulation that highlight the importance of DNA structure in the evolutionary adaptation of promoter sequences.

The role of single nucleotide polymorphisms of miRNAs 100 and 146a as prognostic factors for prostate cancer.

INTRODUCTION: Prostate cancer has a high incidence in men and is the second cause of cancer death among americans male. microRNA (miR) is becoming a potential new prognostic factor for prostate cancer. Single nucleotide polymorphisms (SNPs) are common polymorphisms, characterized by a single exchange of nitrogen based in the DNA. This polymorphism is present in the miRs, altering their function. OBJECTIVE: To evaluate the role of SNP rs1834306 of miR100 and rs2910164 of miR146a in the development and prognosis of prostate cancer. METHODS: One hundred patients diagnosed with prostate cancer and 68 controls were selected. The identification of SNP was rated by quantitative polymerase chain reaction from blood samples, and the analysis was performed within the presence of SNP and the prognostic variables. RESULTS: In the SNP rs1834306 (miR100), a smaller presence of the polymorphic homozygous genotype was identified in patients with PSA >10 ng/mL, (P=0.03); when evaluating only the presence of the polymorphic allele G (P=0.09) it was compared to the presence of the wild type allele A. Among the patients with prostate cancer, SNP rs2910164 (miR146A), the polymorphic allele was more frequent in patients with a Gleason score ⩾7 than in patients with a Gleason score <7, (P=0.043). In patients with prostate cancer, miR100 was overexpressed in those with pT3 staging compared to pT2 and among those who had biochemical recurrence (P = 0.004 and P = 0.011, respectively). CONCLUSIONS: SNP of miR146a acts as a poor prognostic factor (Gleason ⩾7), and the SNP of miR100 is linked to better prognostic data (PSA <10). MiR100 was overexpressed in prostate cancer with worse prognostic factors.

The DNA damage-sensing NER repair factor XPC-RAD23B does not recognize bulky DNA lesions with a missing nucleotide opposite the lesion.

The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were -2.5-3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 ß-hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes.

Response to First Course of Intensified Immunosuppression in Genetically Stratified Steroid Resistant Nephrotic Syndrome.

BACKGROUND AND OBJECTIVES: Intensified immunosuppression in steroid-resistant nephrotic syndrome is broadly applied, with disparate outcomes. This review of patients from the United Kingdom National Study of Nephrotic Syndrome cohort aimed to improve disease stratification by determining, in comprehensively genetically screened patients with steroid-resistant nephrotic syndrome, if there is an association between response to initial intensified immunosuppression and disease progression and/or post-transplant recurrence. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Pediatric patients with steroid-resistant nephrotic syndrome were recruited via the UK National Registry of Rare Kidney Diseases. All patients were whole-genome sequenced, whole-exome sequenced, or steroid-resistant nephrotic syndrome gene-panel sequenced. Complete response or partial response within 6 months of starting intensified immunosuppression was ascertained using laboratory data. Response to intensified immunosuppression and outcomes were analyzed according to genetic testing results, pattern of steroid resistance, and first biopsy findings. RESULTS: Of 271 patients, 178 (92 males, median onset age 4.7 years) received intensified immunosuppression with response available. A total of 4% of patients with monogenic disease showed complete response, compared with 25% of genetic-testing-negative patients (P=0.02). None of the former recurred post-transplantation. In genetic-testing-negative patients, 97% with complete response to first intensified immunosuppression did not progress, whereas 44% of nonresponders developed kidney failure with 73% recurrence post-transplant. Secondary steroid resistance had a higher complete response rate than primary/presumed resistance (43% versus 23%; P=0.001). The highest complete response rate in secondary steroid resistance was to rituximab (64%). Biopsy results showed no correlation with intensified immunosuppression response or outcome. CONCLUSIONS: Patients with monogenic steroid-resistant nephrotic syndrome had a poor therapeutic response and no post-transplant recurrence. In genetic-testing-negative patients, there was an association between response to first intensified immunosuppression and long-term outcome. Patients with complete response rarely progressed to kidney failure, whereas nonresponders had poor kidney survival and a high post-transplant recurrence rate. Patients with secondary steroid resistance were more likely to respond, particularly to rituximab.

Multiplex pyrosequencing quantitative detection combined with universal primer-multiplex-PCR for genetically modified organisms.

A multiplex pyrosequencing quantitative detection technique combined with universal primer-multiplex-PCR (UP-M-PCR) was established. In this study, a pyrosequencing results analysis software was first self-compiled, which realized the DNA sequences degeneration, and converted the pyrosequencing results and base composition of the target sequences into mathematic relations. Five calculation models were put forward based on the actual situation, which adjusted the values smaller than zero or the detection limit. By applying this method, samples containing five genetically modified (GM) lines mixed in random ratio were quantified, it showed that the quantification was very close to the actual value, and the detection sensitivity was as low as 1.47% of a single component, which satisfied most labeling policies. This novel method is realized without fluorescent group labeling, hence the number of targets is not limited by factors inherent in method or equipment, and is proven to be a reliable tool for the quantitative detection.

Cold-Inducible RNA-Binding Protein Prevents an Excessive Heart Rate Response to Stress by Targeting Phosphodiesterase.

RATIONALE: The stress response of heart rate, which is determined by the plasticity of the sinoatrial node (SAN), is essential for cardiac function and survival in mammals. As an RNA-binding protein, CIRP (cold-inducible RNA-binding protein) can act as a stress regulator. Previously, we have documented that CIRP regulates cardiac electrophysiology at posttranscriptional level, suggesting its role in SAN plasticity, especially upon stress conditions. OBJECTIVE: Our aim was to clarify the role of CIRP in SAN plasticity and heart rate regulation under stress conditions. METHODS AND RESULTS: Telemetric ECG monitoring demonstrated an excessive acceleration of heart rate under isoprenaline stimulation in conscious CIRP-KO (knockout) rats. Patch-clamp analysis and confocal microscopic Ca2+ imaging of isolated SAN cells demonstrated that isoprenaline stimulation induced a faster spontaneous firing rate in CIRP-KO SAN cells than that in WT (wild type) SAN cells. A higher concentration of cAMP-the key mediator of pacemaker activity-was detected in CIRP-KO SAN tissues than in WT SAN tissues. RNA sequencing and quantitative real-time polymerase chain reaction analyses of single cells revealed that the 4B and 4D subtypes of PDE (phosphodiesterase), which controls cAMP degradation, were significantly decreased in CIRP-KO SAN cells. A PDE4 inhibitor (rolipram) abolished the difference in beating rate resulting from CIRP deficiency. The mechanistic study showed that CIRP stabilized the mRNA of Pde4b and Pde4d by direct mRNA binding, thereby regulating the protein expression of PDE4B and PDE4D at posttranscriptional level. CONCLUSIONS: CIRP acts as an mRNA stabilizer of specific PDEs to control the cAMP concentration in SAN, maintaining the appropriate heart rate stress response.

Binding of Organometallic Ruthenium Anticancer Complexes to DNA: Thermodynamic Base and Sequence Selectivity.

Organometallic ruthenium(II) complexes [(η6-arene)Ru(en)Cl][PF6] (arene = benzene (1), p-cymene (2), indane (3), and biphenyl (4); en = ethylenediamine) are promising anticancer drug candidates both in vitro and in vivo. In this paper, the interactions between ruthenium(II) complexes and 15-mer single- and double-stranded oligodeoxynucleotides (ODNs) were thermodynamically investigated using high performance liquid chromatography (HPLC) and electrospray ionization mass spectroscopy (ESI-MS). All of the complexes bind preferentially to G8 on the single strand 5'-CTCTCTT7G8T9CTTCTC-3' (I), with complex 4 containing the most hydrophobic ligand as the most reactive one. To the analogs of I (changing T7 and/or T9 to A and/or C), complex 4 shows a decreasing affinity to the G8 site in the following order: -AG8T- (K: 5.74 × 104 M-1) > -CG8C- > -TG8A- > -AG8A- > -AG8C- > -TG8T- (I) ≈ -CG8A- (K: 2.81 × 104 M-1). In the complementary strand of I, the G bases in the middle region are favored for ruthenation over guanine (G) bases in the end of oligodeoxynucleotides (ODNs). These results indicate that both the flanking bases (or base sequences) and the arene ligands play important roles in determining the binding preference, and the base- and sequence-selectivity, of ruthenium complex in binding to the ODNs.

Metagenomics Analysis of Bacterial Population of Tympanosclerotic Plaques and Cholesteatomas.

Objective Chronic otitis media can cause cholesteatomas or tympanosclerosis; however, the pathophysiology of such conditions is not completely known. The aim was to identify a bacterial genome that might be present in tympanosclerotic plaques and cholesteatomas using sequence analysis of the gene responsible for the transcription of 16 ribosomal RNA (rRNA). Study Design Metagenomics analysis of the samples. Setting Samples were collected and evaluated at tertiary care centers. Subjects and Methods Sixty-five tympanosclerotic plaques and 37 cholesteatomas were evaluated. The polymerase chain reaction (PCR) was performed using primers designed for the amplification of the gene responsible for the transcription of bacterial 16 rRNA. The PCR-positive samples were sequenced via Sanger method, and 46 selected samples were analyzed with next-generation sequencing (NGS). Results Sanger sequencing revealed the presence of bacterial genomes in a total of 18 of the 102 samples tested. Sequencing of these genomes indicated the presence of Alloiococcus otitis, Staphylococcus aureus, Achromobacter xylosoxidans, Escherichia coli, Staphylococcus sciuri, Staphylococcus caprae, Parvimonas spp., and Bacillus sp. in the tested samples. The NGS showed 1 or more different bacterial genomes in 44 (95.7%) of the 46 samples tested. Predominately, genome of Clostridiales (27 samples), Staphylococcaceae (24 samples), Peptoniphilaceae (12 samples), and Turicella otitidis (9 samples) were identified. Conclusion The middle ear is inhabited by a diverse microbial community than that previously known. With the use of molecular biology, it has become easier to identify the bacterial genomes and improve our understanding of the role of middle ear microbiota in the pathogenesis of chronic inflammatory ear diseases.

Overview of Next-generation Sequencing Platforms Used in Published Draft Plant Genomes in Light of Genotypization of Immortelle Plant (Helichrysium Arenarium).

INTRODUCTION: Major advancements in DNA sequencing methods introduced in the first decade of the new millennium initiated a rapid expansion of sequencing studies, which yielded a tremendous amount of DNA sequence data, including whole sequenced genomes of various species, including plants. A set of novel sequencing platforms, often collectively named as "next-generation sequencing" (NGS) completely transformed the life sciences, by allowing extensive throughput, while greatly reducing the necessary time, labor and cost of any sequencing endeavor. PURPOSE: of this paper is to present an overview NGS platforms used to produce the current compendium of published draft genomes of various plants, namely the Roche/454, ABI/SOLiD, and Solexa/Illumina, and to determine the most frequently used platform for the whole genome sequencing of plants in light of genotypization of immortelle plant. MATERIALS AND METHODS: 45 papers were selected (with 47 presented plant genome draft sequences), and utilized sequencing techniques and NGS platforms (Roche/454, ABI/SOLiD and Illumina/Solexa) in selected papers were determined. Subsequently, frequency of usage of each platform or combination of platforms was calculated. RESULTS: Illumina/Solexa platforms are by used either as sole sequencing tool in 40.42% of published genomes, or in combination with other platforms - additional 48.94% of published genomes, followed by Roche/454 platforms, used in combination with traditional Sanger sequencing method (10.64%), and never as a sole tool. ABI/SOLiD was only used in combination with Illumina/Solexa and Roche/454 in 4.25% of publications. CONCLUSIONS: Illumina/Solexa platforms are by far most preferred by researchers, most probably due to most affordable sequencing costs. Taking into consideration the current economic situation in the Balkans region, Illumina Solexa is the best (if not the only) platform choice if the sequencing of immortelle plant (Helichrysium arenarium) is to be performed by the researchers in this region.

TRAPR: R Package for Statistical Analysis and Visualization of RNA-Seq Data.

High-throughput transcriptome sequencing, also known as RNA sequencing (RNA-Seq), is a standard technology for measuring gene expression with unprecedented accuracy. Numerous bioconductor packages have been developed for the statistical analysis of RNA-Seq data. However, these tools focus on specific aspects of the data analysis pipeline, and are difficult to appropriately integrate with one another due to their disparate data structures and processing methods. They also lack visualization methods to confirm the integrity of the data and the process. In this paper, we propose an R-based RNA-Seq analysis pipeline called TRAPR, an integrated tool that facilitates the statistical analysis and visualization of RNA-Seq expression data. TRAPR provides various functions for data management, the filtering of low-quality data, normalization, transformation, statistical analysis, data visualization, and result visualization that allow researchers to build customized analysis pipelines.

TRAPR: R Package for Statistical Analysis and Visualization of RNA-Seq Data

High-throughput transcriptome sequencing, also known as RNA sequencing (RNA-Seq), is a standard technology for measuring gene expression with unprecedented accuracy. Numerous bioconductor packages have been developed for the statistical analysis of RNA-Seq data. However, these tools focus on specific aspects of the data analysis pipeline, and are difficult to appropriately integrate with one another due to their disparate data structures and processing methods. They also lack visualization methods to confirm the integrity of the data and the process. In this paper, we propose an R-based RNA-Seq analysis pipeline called TRAPR, an integrated tool that facilitates the statistical analysis and visualization of RNA-Seq expression data. TRAPR provides various functions for data management, the filtering of low-quality data, normalization, transformation, statistical analysis, data visualization, and result visualization that allow researchers to build customized analysis pipelines.

Retention of nucleic acids in ion-pair reversed-phase high-performance liquid chromatography depends not only on base composition but also on base sequence.

The study on nucleic acid retention in ion-pair reversed-phase high-performance liquid chromatography mainly focuses on size-dependence, however, other factors influencing retention behaviors have not been comprehensively clarified up to date. In this present work, the retention behaviors of oligonucleotides and double-stranded DNAs were investigated on silica-based C18 stationary phase by ion-pair reversed-phase high-performance liquid chromatography. It is found that the retention of oligonucleotides was influenced by base composition and base sequence as well as size, and oligonucleotides prone to self-dimerization have weaker retention than those not prone to self-dimerization but with the same base composition. However, homo-oligonucleotides are suitable for the size-dependent separation as a special case of oligonucleotides. For double-stranded DNAs, the retention is also influenced by base composition and base sequence, as well as size. This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase. In addition, no specific influence of guanine and cytosine content was confirmed on retention of double-stranded DNAs. Notably, the space effect resulted from the stereostructure of nucleic acids also influences the retention behavior in ion-pair reversed-phase high-performance liquid chromatography.