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PubMed1999

Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA.

Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.

Base Sequence↗
PubMedDate not supplied

Genetic algorithm for the design of molecules with desired properties.

The design of molecules with desired properties is still a challenge because of the largely unpredictable end results. Computational methods can be used to assist and speed up this process. In particular, genetic algorithms have proved to be powerful tools with a wide range of applications, e.g. in the field of drug development. Here, we propose a new genetic algorithm that has been tailored to meet the demands of de novo drug design, i.e. efficient optimization based on small training sets that are analyzed in only a small number of design cycles. The efficiency of the design algorithm was demonstrated in the context of several different applications. First, RNA molecules were optimized with respect to folding energy. Second, a spinglass was optimized as a model system for the optimization of multiletter alphabet biopolymers such as peptides. Finally, the feasibility of the computer-assisted molecular design approach was demonstrated for the de novo construction of peptidic thrombin inhibitors using an iterative process of 4 design cycles of computer-guided optimization. Synthesis and experimental fitness determination of only 600 different compounds from a virtual library of more than 10(17) molecules was necessary to achieve this goal.

Algorithms↗
PubMed2025

Next-generation sequencing in breast cancer: current clinical applications and future directions.

INTRODUCTION: Breast cancer is a heterogeneous disease that claims 670,000 lives by 2022. Omic technologies, particularly next generation sequencing (NGS) offers promising avenues for precision medicine. American Society of Clinical Oncology (ASCO) outlines genomic testing's utility, emphasizing prognostic and diagnostic potential. OBJECTIVES: This review succinctly explores NGS's evolution and clinical applications of NGS in breast cancer, thereby guiding future research to enhance patient care. METHODS: Comprehensive literature searches were conducted using databases such as PubMed, Google Scholar, and ResearchGate, focusing on keywords including breast cancer, HER-2 low breast cancer, circulating tumour DNA, single-cell RNA sequencing, and next-generation sequencing. Peer-reviewed, high-quality articles published in English were selected for inclusion. RESULTS: Previous studies have explored the evolution of NGS technology and its clinical applications in breast cancer, including genomic and transcriptomic characterization, treatment guidance, and resistance prediction. Molecular profiling of challenging entities such as early-onset breast cancer and HER-2 low tumours was summarized, with key findings highlighted. This review also discusses emerging technologies, including circulating DNA and single-cell sequencing, as promising avenues for discovery. CONCLUSION: NGS has revealed the genomic and transcriptomic diversity of breast cancer, identifying actionable alterations associated with chemotherapy response and resistance to therapies such as trastuzumab, TKIs, and CDK4/6 inhibitors. Circulating tumour DNA (ctDNA) shows potential for diagnosis, prediction, prognosis, and monitoring, despite tumour heterogeneity. Single-cell analysis enables exploration of individual cell transcriptomes, though high costs and low throughput remain barriers to widespread adoption. HER2-low tumours continue to pose significant research challenges.

Humans↗
PubMed2003

Development of functional human monoclonal single-chain variable fragment antibody against HIV-1 from human cervical B cells.

A panel of novel recombinant single-chain variable fragment (scFv) antibody against human immunodeficiency virus type-1 (HIV-1) was isolated and characterized. We generated human scFvs using RNA harvested from cervical B lymphocytes of Kenyan prostitutes who are highly exposed to HIV-1, but remain persistently seronegative. The variable regions of the heavy (VH) and light (VL) chain antibody genes were selected as hybrids using guided-selection with the VL and VH, respectively, of a derivative of IgGb(12) using the phagemid vector pComb3X. IgGb(12) is a previously well-characterized HIV-1 neutralizing human monoclonal antibody (MAb). One of the hybrid scFv, IgA6/4L, neutralizes HIV-1 infectivity in in vitro cell culture assay. The cervical VH and VL chain antibody genes were connected by a DNA linker and subcloned in pComb3X. The cervical scFv clones were functional in recognizing HIV-1 gp120 by enzyme-linked immunosorbant assay (ELISA) and on cells in flow cytometry. Whole IgGb(12) does not inhibit binding of clones IgA6/5k nor IgA6/30lambda to gp120, which suggests that they bind different epitopes. Nucleotide sequence analysis of the cervical scFv show the clones are unique and reveal interesting characteristics of human cervical V gene pools. This work demonstrates, for the first time, cloning of a functional scFv MAb to a sexually transmitted disease pathogen from local cervical B-cell pools in exposed humans.

Amino Acid Sequence↗
PubMed2002

ARGONAUTE1 is required for efficient RNA interference in Drosophila embryos.

Double-stranded RNA (dsRNA) triggers homology-dependent posttranscriptional gene interference (RNAi) in a diverse range of eukaryotic organisms, in a process mechanistically related to viral and transgene-mediated cosuppression. RNAi is characterized by the conversion of long dsRNA into approximately 21-25-nt small interfering RNAs (siRNA) that guide the degradation of homologous mRNA. Many of the genes required for siRNA production and target mRNA degradation are widely conserved. Notably, members of the Argonaute-like gene family from Arabidopsis, Caenorhabditis elegans, Drosophila, and Neurospora have been genetically and/or biochemically identified as components of the RNAi/cosuppression pathway. We show here that mutations in the Drosophila Argonaute1 (AGO1) gene suppress RNAi in embryos. This defect corresponds to a reduced ability to degrade mRNA in response to dsRNA in vitro. Furthermore, AGO1 is not required for siRNA production in vitro nor can the introduction of siRNA bypass AGO1 mutants in vivo. These data suggest that AGO1 functions downstream of siRNA production.

ATP-Binding Cassette Transporters↗
PubMed2005

HPLC analysis of plant DNA methylation: a study of critical methodological factors.

HPLC analysis of nucleosides is important for determining total DNA methylation in plants and can be used to help characterise epigenetic changes during stress, growth and development. This is of particular interest for in vitro plant cultures as they are highly susceptible to genetic change. HPLC methodologies have been optimised for mammalian and microbial DNA, but not for plants. This study examines critical methodological factors in the HPLC analysis of plant DNA methylation using in vitro cultures of Ribes ciliatum. HPLC revealed that complete removal of RNA from plant DNA extractions is difficult using RNase (A and T1) digestions and LiCl precipitation. This suggests that base analysis should be avoided when using these RNA removal techniques, as bases from residual RNA fragments will inflate peak areas for DNA-derived bases. Nucleoside or nucleotide analysis is therefore recommended as a more suitable option as RNA and DNA constituents can be readily separated. DNA digestion was also a critical factor as methylation was under-estimated following incomplete nuclease digestion and over-estimated following incomplete phosphatase digestion. The units of enzyme required for complete DNA digestion was optimised and found to be 20-200 times less for nuclease P1 and 15 times less for alkaline phosphatase as compared with previous protocols. Digestion performance was conveniently monitored using marker peaks that indicate incomplete digestion products. This study identifies critical components of HPLC analysis and offers a comprehensive guide for the stringent analysis of DNA methylation in plants.

Chromatography, High Pressure Liquid↗
PubMed1985

Sequence comparison of the rDNA introns from six different species of Tetrahymena.

We have studied the sequence variation of the rDNA intron among six species of Tetrahymena. From these data, the intron appears to be relatively well conserved in evolution. We have evaluated the sequence variations among the most distant of these species in relation to the secondary structure model for the intron RNA of Cech et al. (Proc. Natl. Acad. Sci. U.S.A. 80, 3903 (83)). Most of the sequence variation in the four new sequences reported here is found in single stranded loops in the model. However, in four cases we found nucleotide substitutions in duplex stem regions, two of them involving compensating base pair changes. Interestingly, one of these is found in a region that is known to be dispensable in the in vitro splicing reaction suggesting differences between the in vivo and in vitro reactions. One of the single nucleotide deletions is found in the so-called "internal guide sequence" which has been implicated in the alignment process during splicing. In conclusion, none of the observed natural sequence variations are in disfavor of the proposed secondary structure model.

Base Sequence↗
PubMed2004

Global effects of human papillomavirus type 18 E6/E7 in an organotypic keratinocyte culture system.

The effects of human papillomavirus type 18 (HPV-18) E6 and E7 proteins on global patterns of host gene expression in primary human keratinocytes grown in organotypic raft culture system were assessed. Primary human keratinocytes were infected with retroviruses that express the wild-type HPV-18 E6 and E7 genes from the native differentiation-dependent HPV enhancer-promoter. Total RNA was isolated from raft cultures and used to generate probes for querying Affymetrix U95A microarrays, which contain >12,500 human gene sequences. Quadruplicate arrays of each E6/E7-transduced and empty vector-transduced samples were analyzed by 16 pairwise comparisons. Transcripts altered in > or =12 comparisons were selected for further analysis. With this approach, HPV-18 E6/E7 expression significantly altered the expression of 1,381 genes. A large increase in transcripts associated with DNA and RNA metabolism was observed, with major increases noted for transcription factors, splicing factors, and DNA replication elements, among others. Multiple genes associated with protein translation were downregulated. In addition, major alterations were found in transcripts associated with the cell cycle and cell differentiation. Our study provides a systematic description of transcript changes brought about by HPV-18 E6/E7 in a physiologically relevant model and should furnish a solid source of information to guide future studies.

Cell Culture Techniques↗
PubMed2006

Role of individualization of hepatitis C virus (HCV) therapy duration in HIV/HCV-coinfected individuals.

OBJECTIVE: The aim of this study was to assess the efficacy, safety and tolerability of pegylated interferon and ribavirin in HIV/hepatitis C virus (HCV)-coinfected patients, prescribed for the same duration and at the same dosage as that used in HCV monoinfection studies. DESIGN: It was an open-label, single-centre, prospective study. METHODS: Forty-five patients coinfected with HIV and HCV with CD4 counts >200 cells/microL were treated with pegylated interferon-alpha2b 1.5 microg/kg/week and ribavirin 1000-1200 mg/day for 24-48 weeks depending on HCV genotype. Safety and tolerability were assessed weekly for the first month and monthly thereafter. Virological response was assessed at weeks 4, 12 and 24 and at the end of treatment and 12 and 24 weeks post completion of treatment. The primary endpoint was defined as undetectable HCV RNA at 24 weeks post completion of treatment [sustained virological response (SVR)]. RESULTS: The majority of patients were male and had been injecting drug users. Sixty per cent were on antiretroviral therapy. In an intention-to-treat analysis, 53% had an SVR (genotype 1, 19% and genotype 2/3, 75%). All patients who had undetectable HCV RNA at week 4 of HCV treatment [very early virological response (VEVR)] had a SVR. On multivariate analysis only HCV genotype predicted SVR. Adverse events occurred frequently. CONCLUSIONS: These results indicate that 24 weeks of HCV treatment may be adequate for HIV-infected individuals coinfected with HCV genotype 2 or 3. VEVR can predict SVR in this group and may be used to guide the subgroup of genotype 2/3 individuals who will respond to 24 weeks of treatment.

Adult↗
PubMed2004

Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method.

In acute myeloid leukaemia (AML), alterations in apoptotic pathways are crucial for treatment outcome, resulting either in refractoriness or in minimal residual disease (MRD). The apoptosis characteristics of MRD cells may differ from those at diagnosis and thereby determine the adequacy of further treatment. Such characteristics are largely unknown, since studies hereto are hampered by minimal cell availability. This study explores the applicability of the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) for gene expression analysis of small amounts of RNA obtained from MRD cells. Reproducibility and dilution experiments showed that the relative expression of 37 apoptosis-related genes starting with only 1000 cells could be measured with 12% variation; for 100 cells, 31/37 genes could still be quantified, though expression variation increased. In practice 100-1000 leukemic cells can be obtained from bone marrow samples with clinically relevant MRD percentages of 0.01-0.1. Procedures often necessary to obtain AML blasts, that is, FACS-sorting, freeze-thawing or combinations are possible, provided that selected viable nonapoptotic cells are used. Concluding, RT-MLPA allows accurate gene expression profiling of MRD cells. This method will help to gain insight into the processes of MRD emergence and persistence in AML, which may ultimately guide new therapeutic strategies in AML.

Acute Disease↗
PubMed1985

Sites of circularization of the Tetrahymena rRNA IVS are determined by sequence and influenced by position and secondary structure.

The sequence of the cloned Tetrahymena ribosomal RNA intervening sequence (IVS) was altered at the site to which circularization normally occurs. The alterations caused circularization to shift to other sites, usually a nearby position which followed three pyrimidines. While a tripyrimidine sequence was the major determinant of a circularization site, both location of a sequence and local secondary structure may influence the use of that sequence. For some constructs circularization appeared to occur at the position following the 5' G, the nucleotide added to the IVS during its excision. Portions of the internal guide sequence (IGS), proposed to interact with the 3'exon were deleted without preventing exon ligation. Thus if the IGS-3'exon interaction exists, it is not essential for splicing in vitro.

Animals↗
PubMed2006

Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing.

Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended ("off-target") silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence-independent chemical modifications that reduced silencing of partially complementary transcripts by all siRNAs tested. Silencing of perfectly matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2'-O-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position dependence of 2'-O-methyl ribosyl modification contrasts with the broader position dependence of base-pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts.

Base Sequence↗
PubMed2005

Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome.

Initiation of protein synthesis is a universally conserved event that requires initiation factors IF1, IF2 and IF3 in prokaryotes. IF2 is a GTPase essential for binding initiator transfer RNA to the 30S ribosomal subunit and recruiting the 50S subunit into the 70S initiation complex. We present two cryo-EM structures of the assembled 70S initiation complex comprising mRNA, fMet-tRNA(fMet) and IF2 with either a non-hydrolyzable GTP analog or GDP. Transition from the GTP-bound to the GDP-bound state involves substantial conformational changes of IF2 and of the entire ribosome. In the GTP analog-bound state, IF2 interacts mostly with the 30S subunit and extends to the initiator tRNA in the peptidyl (P) site, whereas in the GDP-bound state IF2 steps back and adopts a 'ready-to-leave' conformation. Our data also provide insights into the molecular mechanism guiding release of IF1 and IF3.

Cryoelectron Microscopy↗
PubMed2026

Beyond blacklists: a critical assessment of exclusion set generation strategies and alternative approaches.

MOTIVATION: Short-read sequencing data can be affected by alignment artifacts in certain genomic regions. Removing reads overlapping these exclusion regions, previously known as Blacklists, help to potentially improve biological signal. Alternatively, "sponge" or decoy sequences have been proposed to reduce alignment artifacts. RESULTS: We examined the widely used Blacklist software and found that pre-generated exclusion sets were difficult to reproduce due to sensitivity to input data, aligner choice, and read length. We further explored the use of "sponge" sequences-unassembled genomic regions such as satellite DNA, ribosomal DNA, and mitochondrial DNA-as an alternative approach. We additionally investigated the effect of the T2T-CHM13 genome assembly on improving biological signals. Aligning reads to a genome that includes sponge sequences reduced signal correlation in ChIP-seq data comparably to Blacklist-derived exclusion sets while preserving biological signal. Sponge-based alignment also had minimal impact on RNA-seq gene counts, suggesting broader applicability beyond chromatin profiling. These results highlight the limitations of fixed exclusion sets, and recommend the use of the T2T-CHM13 assembly or, for the hg38 genome assembly, "sponge" sequences as an alignment-guided strategy for reducing artifacts and improving functional genomics analyses.

Software↗
PubMed2004

Biology of chronic myeloid leukemia and possible therapeutic approaches to imatinib-resistant disease.

Chronic myeloid leukemia (CML) is a stem cell disorder caused by a constitutively activated tyrosine kinase, the Bcr-Abl oncoprotein. An inhibitor of this tyrosine kinase, imatinib mesylate, is rapidly becoming the first-line therapy for CML. However, the development of resistance to this drug is a frequent setback, particularly in patients in advanced phases of the disease. Several mechanisms of resistance have been described, the most frequent of which are amplification and/or mutations of the BCR-ABL gene. To overcome resistance, several approaches have been studied in vitro and in vivo. They include dose escalation of imatinib, combination of imatinib with chemotherapeutic drugs, alternative Bcr-Abl inhibitors, inhibitors of kinases downstream of Bcr-Abl, farnesyl and geranylgeranyl transferase inhibitors, histone deacetylase, proteasome and cyclin-dependent kinase inhibitors, arsenic trioxide, hypomethylating agents, troxacitabine, targeting Bcr-Abl messenger RNA, and immunomodulatory strategies. It is important to understand that these approaches differ in efficiency, which is often dependent on the mechanisms of resistance. Further investigations into the molecular mechanisms of disease and how to specifically target the abnormal processes will guide the design of new treatment modalities in future clinical trials.

Antineoplastic Agents↗
PubMed2002

[Swedish guidelines for antiretroviral treatment of HIV. Good compliance is crucial for a good result].

Antiretroviral combination therapy has markedly decreased the mortality and morbidity in HIV-disease during the last years. An increasing number of new antiretroviral agents has been introduced and there has been a rapid evolution of new information regarding treatment of HIV. Physicians and patients have to deal with medical associated side-effects, problems with adherence, and viral resistance. Recently a national expert group has prepared guidelines for antiretroviral treatment in HIV-infected adults and adolescents which are summarized in this report. The Swedish guidelines recommend initiation of treatment when the CD4-cell-count is in the range between 200 and 350 x 10(6)/l in asymptomatic HIV-infection. They emphasize that the patient should be carefully prepared before treatment initiation to avoid adherence difficulties which regularly leads to viral failure. As first line is suggested a therapy with 2 NRTIs (nucleoside reverse transcriptase inhibitors) in combination with either a NNRTI (non-nucleoside RTI) or a protease inhibitor. When protease inhibitors are chosen, ritonavir-boosted combinations are most often to prefer. The virological treatment goal is to achieve a decrease in HIV-RNA with 1.5-2 log after 4 weeks and to below 50 copies/ml after 3-4 months of therapy. If a therapy fails, the causes should be carefully investigated for adherence difficulties, medicine interactions, and viral resistance. Treatment change should be guided by a thorough drug treatment history and the results of resistance testing. Treatment of HIV is complex and it is recommended that it should be conducted by an expert in the field.

Adolescent↗
PubMed2001

Binding enhancement by tertiary interactions and suicide inhibition of a Candida albicans group I intron by phosphoramidate and 2'-O-methyl hexanucleotides.

Candida albicans is one of many infectious pathogens that are evolving resistance to current treatments. RNAs provide a large class of targets for new therapeutics for fighting these organisms. One strategy for targeting RNAs uses short oligonucleotides that exhibit binding enhancement by tertiary interactions in addition to Watson-Crick pairing. A potential RNA target in C. albicans is the self-splicing group I intron in the LSU rRNA precursor. The recognition elements that align the 5' exon splice site for a ribozyme derived from this precursor are complex [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. These recognition elements have been used to guide design of hexanucleotide mimics of the 5' exon that have backbones modified for nuclease stability. These hexanucleotides bind as much as 100000-fold more tightly to a ribozyme derived from the intron than to a hexanucleotide mimic of the intron's internal guide sequence, r(GGAGGC). Several of these oligonucleotides inhibit precursor self-splicing via a suicide inhibition mechanism. The most promising suicide inhibitor is the ribophosphoramidate rn(GCCUC)rU, which forms more trans-spliced than cis-spliced product at oligonucleotide concentrations of >100 nM at 1 mM Mg(2+). The results indicate that short oligonucleotides modified for nuclease stability can target catalytic RNAs when the elements of tertiary interactions are complex.

Binding, Competitive↗
PubMed2002

Identification of tandemly-repeated C/D snoRNA genes at the imprinted human 14q32 domain reminiscent of those at the Prader-Willi/Angelman syndrome region.

A human imprinted domain at 14q32 contains two co-expressed and reciprocally imprinted genes, DLK1 and GTL2, which are expressed from the paternally and maternally inherited alleles, respectively. We have previously shown that another imprinted locus, on human 15q11-q13, contains a large number of tandemly repeated C/D small nucleolar RNA genes (or C/D snoRNAs) only expressed from the paternal allele. Here we show that the region downstream from the GTL2 gene is also characterized by a transcription unit spanning many repeated intron-encoded C/D snoRNA genes, most of them arranged into two tandem arrays of 31 and 9 copies. Intriguingly, these snoRNAs depart from previously reported rRNA or snRNA methylation guides by their tissue-specific expression and by their lack of complementarity to rRNA or snRNA within their sequences. Analysis of the orthologous region in the mouse shows that the previously reported maternally expressed Rian gene, located downstream of Gtl2 on the distal 12 chromosome, encodes at least nine C/D snoRNAs. Through a systematic search in rodents, we could identify other C/D snoRNA genes in this domain. All snoRNAs identified on mouse distal 12 are brain-specific and only expressed from the maternally inherited allele. The human imprinted 14q32 domain therefore shares common genomic features with the imprinted 15q11-q13 loci. This link between tandemly repeated C/D snoRNA genes and genomic imprinting suggests a role for these snoRNAs and/or their host non-coding RNA genes in the evolution and/or mechanism of the epigenetic imprinting process.

Angelman Syndrome↗